Non-mass determined base compositions for nucleic acid detection

ABSTRACT

The present invention provides systems, methods, and compositions for nucleic acid detection based on non-mass determined base compositions. For example, in certain embodiments, base count data for a template nucleic acid is generated using an approach that does not measure molecular mass of the template nucleic acid (e.g., by sequencing the template nucleic acid) and a database comprising base count entries is queried to identify the target nucleic acid. In particular embodiments, sequencing is employed which is conducted in substantially real-time.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 13/389,139 filed Apr. 4, 2012, which is a national phaseapplication under 35 U.S.C. §371 of PCT International Application No.PCT/US2010/044774, filed on 6 Aug. 2010, which claims priority to U.S.Provisional Patent Application Ser. No. 61/231,907 filed Aug. 6, 2009,each of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to systems, methods, and compositions fornucleic acid detection based on non-mass determined base compositions.For example, in certain embodiments, base count data for a templatenucleic acid is generated using an approach that does not measuremolecular mass of the template nucleic acid (e.g., by sequencing thetemplate nucleic acid) and a database comprising base count entries isqueried to identify the target nucleic acid. In particular embodiments,sequencing is employed which is conducted in substantially real-time.

BACKGROUND OF THE INVENTION

In the United States, hospitals report well over 5 million cases ofrecognized infectious disease-related illnesses annually. Significantlygreater numbers remain undetected, both in the inpatient and communitysetting, resulting in substantial morbidity and mortality. Criticalintervention for infectious disease relies on rapid, sensitive andspecific detection of the offending pathogen, and is central to themission of microbiology laboratories at medical centers. Unfortunately,despite the recognition that outcomes from infectious illnesses aredirectly associated with time to pathogen recognition, as well asaccurate identification of the class and species of microbe, and abilityto identify the presence of drug resistance isolates, conventionalhospital laboratories often remain encumbered by traditional slowmulti-step culture based assays. Other limitations of the conventionallaboratory which have become increasingly apparent include: extremelyprolonged wait-times for pathogens with long generation time (up toseveral weeks); requirements for additional testing and wait times forspeciation and identification of antimicrobial resistance; diminishedtest sensitivity for patients who have received antibiotics; andabsolute inability to culture certain pathogens in disease statesassociated with microbial infection.

For more than a decade, molecular testing has been heralded as thediagnostic tool for the new millennium, whose ultimate potential couldinclude forced obsolescence of traditional hospital laboratories.However, despite the fact that significant advances in clinicalapplication of nucleic acid amplification techniques have occurred, thepracticing physician still relies principally on standard techniques,such as culturing. As such, what is needed are rapid sensitivediagnostics systems and methods.

SUMMARY OF THE INVENTION

The present invention provides systems, methods, and compositions fornucleic acid detection based on non-mass determined base compositions.For example, in certain embodiments, base count data for a templatenucleic acid is generated using an approach that does not measuremolecular mass of the template nucleic acid (e.g., by sequencing thetemplate nucleic acid) and a database comprising base count entries isqueried to identify the target nucleic acid. Essentially any sequencingapproach is optionally utilized in the present invention, including, forexample, pyrosequencing, zero-mode waveguide type sequencing, nanoporesequencing, and the like. In particular embodiments, sequencing isemployed which is conducted in substantially real-time.

In some embodiments, the present invention provides methods ofidentifying a template nucleic acid comprising: (a) determining at leasta partial base count (e.g., a partial base count or a full base count)of at least a subsequence (e.g., a sub-sequence or a full sequence) ofat least one template nucleic acid and/or a complement thereof, using anapproach that does not measure molecular mass (e.g., without using massspectrometry type methods) of the template nucleic acid, to produce basecount data; and (b) querying a database comprising at least one basecount entry (e.g., comprising 1 . . . 5 . . . 10 . . . 100 . . . 1000 .. . 10,000 . . . 50,000 . . . or more base count entries) correspondingto an identified nucleic acid to produce a match of the base count datawith the base count entry, thereby identifying the template nucleicacid.

In certain embodiments, the base count or partial base count isdetermined by sequencing. In other embodiments, the base count orpartial base count is determined by liquid chromatography, such as HighPerformance Liquid Chromatography (HPLC). In further embodiments, thebase count or partial base count is determined by paper chromatography(e.g., 2-D paper chromatography).

In particular embodiments, at least a partial base count only includesthe number of A's present in a template nucleic acid (e.g., A₁₄). Inparticular embodiments, at least a partial base count only includes thenumber of C's present in a template nucleic acid (e.g., C₁₇). Inparticular embodiments, at least a partial base count only includes thenumber of G's present in a template nucleic acid (e.g., G₁₀). Inparticular embodiments, at least a partial base count only includes thenumber of T's present in a template nucleic acid (e.g., T₁₉). In otherembodiments, at least a partial base count only includes two of thecanonical bases (e.g., A₁₄, C₁₇). In further embodiments, at least apartial base count only includes three of the canonical bases (e.g.,A₁₄, C₁₇, G₁₀). In additional exemplary embodiments, at least a partialbase count includes all four canonical bases (e.g., A₁₄, C₁₇, G₁₀, T₁₉).

In certain embodiments, the template nucleic acid comprises DNA or RNA.In other embodiments, the determining is performed in real-time orsubstantially real-time (e.g., using a device comprising a zero-modewaveguide). In particular embodiments, the template nucleic acidcomprises a mammalian nucleic acid, a bacterial nucleic acid, a viralnucleic acid, a fungal nucleic acid, or a protozoal nucleic acid. Infurther embodiments, the template nucleic acid is attached to a solidsupport. In additional embodiments, the methods further compriseamplifying the template nucleic acid prior to or during (a). In certainembodiments, the method further comprise obtaining the template nucleicacid from one or more sample sources selected from the group consistingof: an environmental sample and a sample derived from a subject.

In particular embodiments, step (a) comprises contacting the templatenucleic acid with at least one nucleotide incorporating biocatalyst,labeled nucleotides, and at least one primer nucleic acid that is atleast partially complementary to at least a subsequence of the templatenucleic acid, under conditions whereby the nucleotide incorporatingbiocatalyst extends the primer nucleic acid to produce an extendedprimer nucleic acid by incorporating the labeled nucleotides at aterminal end of the extended primer nucleic acid, wherein nucleotidesthat comprise different nucleobases comprise different labels, whereinthe different labels produce detectable signals as or after the labelednucleotides are incorporated at the terminal end of the extended primernucleic acid, which detectable signals identify the labeled nucleotidesincorporated at the terminal end of the extended primer nucleic acidand/or complementary nucleotides in the template nucleic acid, andwherein the detectable signals are detected as or after the labelednucleotides are incorporated at the terminal end of the extended primernucleic acid to thereby determine the base count of the subsequence ofthe template nucleic acid and/or the complement thereof. In otherembodiments, the labels comprise different fluorescent labels andwherein the detectable signals are detected using a fluorescencemicroscope. In some embodiments, the at least one primer nucleic acid isa primer pair, wherein the primer pair is configured to hybridize withconserved regions of the two or more different bioagents and flankvariable regions of the two or more different bioagents.

In particular embodiments, the terminal end of the extended primernucleic acid is the 3′ terminal end. In further embodiments, thenucleotide incorporating biocatalyst comprises an enzyme selected fromthe group consisting of: a polymerase, a terminal transferase, a reversetranscriptase, a polynucleotide phosphorylase, and a telomerase. Inadditional embodiments, the nucleotide incorporating biocatalystcomprises one or more modifications. In some embodiments, the nucleotideincorporating biocatalyst is an enzyme derived from an organism that isselected from the group consisting of: Thermus antranikianii, Thermusaquaticus, Thermus caldophilus, Thermus chliarophilus, Thermusfiliformis, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermusoshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermussilvanus, Thermus species Z05, Thermus species sps 17, Thermusthermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Anaerocellum thermophilum, Bacillus caldotenax, and Bacillusstearothermophilus.

In certain embodiments, the nucleotide incorporating biocatalystcomprises a Φ29 DNA polymerase. In other embodiments, a label isattached to one of a heterocyclic base of a labeled nucleotide, a sugarmoiety of a labeled nucleotide, and a phosphate group of a labelednucleotide. In further embodiments, a linker attaches a label to alabeled nucleotide. In particular embodiments, the extended primernucleic acid is complementary to a subsequence of the template nucleicacid. In further embodiments, the extended primer nucleic acid iscomplementary to a full-length sequence of the template nucleic acid. Incertain embodiments, the primer nucleic acid comprises an intelligentprimer. In other embodiments, the label comprises a fluorescent dye, anon-fluorescent label, a colorimetric label, a chemiluminescent label, abioluminescent label, a radioisotope, an antibody, an antigen, biotin, ahapten, or an enzyme. In other embodiments, the label is a fluorescentdye selected from the group consisting of: a rhodamine dye, afluorescein dye, a halofluorescein dye, a dichlororhodamine dye, anenergy transfer dye, a Lucifer dye, Oregon Green, and a cyanine dye. Insome embodiments, the label is a fluorescent dye selected from the groupconsisting of: JOE, VIC, TET, HEX, PAM, R6G, R110, TAMRA, and ROX. Inparticular embodiments, the label is a radioisotope selected from thegroup consisting of: ³H, ¹⁴C, ²²Na, ³²P, ³³P, ³⁵S, ⁴²K, ⁴⁵Ca, ⁵⁹Fe,¹²⁵I, and ²⁰³Hg.

In certain embodiments, the methods comprise determining at leastpartial base counts of subsequences of two or more template nucleicacids and/or complements thereof, using an approach that does notmeasure molecular mass of the template nucleic acids to produce the basecount data. In other embodiments, the methods comprise querying thedatabase to produce two or more matches of the base count data with twoor more base count entries to thereby identify the template nucleicacid. In other embodiments, identifying the template nucleic acidcomprises identifying an organismal source of the template nucleic acidand/or one or more properties of the template nucleic acid. In someembodiments, the properties of the template nucleic acid comprise agenotype, a virulence property, a pathogenic property, and/or a drugresistance property. In particular embodiments, the determining isperformed in substantially real-time.

In other embodiments, the organismal source comprises a mammal, abacterium, a virus, a fungus, or a protozoan. In further embodiments,the organismal source is identified at one or more taxonomic rank levelsselected from the group consisting of: a Domain, a Superphylum, aSuperdivision, a Superclass, a Superorder, a Superfamily, aSuperspecies, a Kingdom, a Phylum, a Division, a Class, a Legion, anOrder, a Family, a Tribe, a Genus, a Species, a Subkingdom, a Subphylum,a Subclass, a Cohort, a Suborder, a Subfamily, a Subtribe, a Subgenus, aSubspecies, an Infrakingdom, a Branch, an Infraphylum, an Infraclass, anInfraorder, an Alliance, an Infraspecies, a Microphylum, a Pan/class,and a Parvorder.

In some embodiments, the present invention provides systems comprising:at least one sequencing device; at least one primer pair configured tohybridize with conserved regions of two or more different bioagents andflank variable regions of the two or more different bioagents; and acomputer readable medium having one or more logic instructions fordirecting a system to: (a) determine at least a partial base count of atleast a subsequence of at least one template nucleic acid and/or acomplement thereof, using an approach that does not measure molecularmass of the template nucleic acid, to produce base count data; and (b)query a database comprising at least one base count entry correspondingto an identified nucleic acid to produce a match of the base count datawith the base count entry to thereby identifying the template nucleicacid.

In certain embodiments, the system comprise instructions for determiningat least a partial base count of at least a subsequence of at least onetemplate nucleic acid and/or a complement thereof, in substantiallyreal-time without measuring the molecular mass of the template nucleicacid to produce base count data using the zero-mode waveguide and theprimer nucleic acid and for identifying the template nucleic acid usingthe base count data. In certain embodiments, the sequencing devicecomprises a zero-mode waveguide. In other embodiments, the systemscomprise at least one nucleotide incorporating biocatalyst and/orlabeled nucleotides. In further embodiments, the systems furthercomprise one or more containers for packaging the zero-mode waveguide,or the primer pair.

In certain embodiments, the present invention provides a computerprogram product, comprising a computer readable medium having one ormore logic instructions for directing a system to: (a) determine atleast a partial base count of at least a subsequence of at least onetemplate nucleic acid and/or a complement thereof (e.g., insubstantially real-time), using an approach that does not measuremolecular mass of the template nucleic acid, to produce base count data;and (b) query a database comprising at least one base count entrycorresponding to an identified nucleic acid to produce a match of thebase count data with the base count entry to thereby identifying thetemplate nucleic acid.

In some embodiments, the present invention provides system comprising: areaction vessel or substrate; a detector configured to detect detectablesignals produced in or on the reaction vessel or substrate, whichdetectable signals correspond to at least some nucleobases incorporatedinto a nucleic acid to generate nucleobase incorporation data; adatabase of base count entries indexed to identified nucleic acids; anda controller configured to correlate the nucleobase incorporation datawith detected base counts and to query the database for a match betweenthe detected base counts and the base count entries to thereby identifyor detect the nucleic acids.

In particular embodiments, the reaction vessel or substrate comprises atleast one zero-mode waveguide. In further embodiments, the detectorcomprises a fluorescence microscope. In other embodiments, the systemscomprise at least one material transfer component that transfersmaterial to and/or from the reaction vessel or substrate. In otherembodiments, the systems comprise at least one thermal modulatorconfigured to modulate temperature in the reaction vessel or substrate.In other embodiments, the reaction vessel or substrate comprises atleast one primer nucleic acid is selected from the primer nucleic acidsdisclosed in one or more of the published documents incorporated byreference herein. In other embodiments, the systems comprise at leastone nucleic acid sample preparation component and/or at least onenucleic acid amplification component.

In further embodiments, the present invention provides methods ofidentifying a target nucleic acid in a sample comprising: (a) at leastpartially sequencing the target nucleic acid, or amplified sequencesthereof, to generate sequence data; (b) determining at least a partialbase count from the sequence data to generate base count data; and (c)querying a database comprising at least one base count entrycorresponding to the target nucleic acid to produce a match of the basecount data with the base count entry, thereby identifying the targetnucleic acid.

In some embodiments, the present invention provides methods ofidentifying a target nucleic acid in a sample comprising: (a) amplifyingone or more segments of the nucleic acid in the sample using at leastone purified oligonucleotide primer pair to generate amplifiedsequences, wherein the primer pair is configured to hybridize withconserved regions of two or more different bioagents and flank variableregions of the two or more different bioagents; (b) at least partiallysequencing the amplified sequences to generate sequence data; (c)determining at least a partial base count from the sequence data togenerate base count data; and (d) querying a database comprising atleast one base count entry corresponding to the target nucleic acid toproduce a match of the base count data with the base count entry,thereby identifying the target nucleic acid.

In certain embodiments, the molecular mass of the target sequence or theamplified sequences is not determined in the identifying the targetnucleic acid. In further embodiments, the sequencing is performed inreal-time or substantially real-time. In other embodiments, theidentifying the target nucleic acid is performed in substantiallyreal-time. In further embodiments, the at least partially sequencing thetarget nucleic acid is performed with a device comprising at least onezero-mode waveguide. In some embodiments, the primer pair comprisesforward and reverse primers that are about 20 to 35 nucleobases inlength.

In other embodiments, the present invention provides systems comprising:(a) a sequencing device configured to generate nucleic acid sequencedata corresponding to the nucleic acid sequence of one or more ampliconsproduced using at least one purified oligonucleotide primer pair thatcomprises forward and reverse primers, wherein the primer pair comprisesnucleic acid sequences that are substantially complementary to nucleicacid sequences of two or more different bioagents; and (b) a controlleroperably connected to the sequencing device, the controller configuredto: i) determine at least a partial base count from the nucleic acidsequence data to generate base count data, and ii) query a database withthe base count data, wherein the database comprises at least one basecount entry corresponding to a target nucleic acid.

In certain embodiments, the database comprises base compositioninformation (entries) for at least three different bioagents. In otherembodiments, the database comprises base composition information for atleast 2 . . . 10 . . . 50 . . . 100 . . . 1000 . . . 10,000, or 100,000different bioagents. In some embodiments, the base composition datacomprises at least 10 . . . 50 . . . 100 . . . 500 . . . 1000 . . . 1000. . . 10,000 . . . or 100,000 unique base compositions. In furtherembodiments, the database is stored on a local computer. In particularembodiments, the database is accessed from a remote computer over anetwork. In further embodiments, the base composition information in thedatabase is associated with bioagent identity. In certain embodiments,the base composition data in the database is associated with bioagentgeographic origin.

In some embodiments, the present invention provides methods ofidentifying a target nucleic acid in a sample comprising: (a) amplifyingone or more segments of the target nucleic acid in the sample using atleast one purified oligonucleotide primer pair to generate a samplecomprising amplified sequences, wherein said primer pair comprises aforward primer and a reverse primer and is configured to hybridize withconserved regions of two or more different bioagents and flank variableregions of the two or more different bioagents; (b) treating the samplewith reagents such that the amplified sequences in the sample aredigested into monodeoxynucleosides; (c) quantitating themonodeoxynucleosides using HPLC, paper chromatography, or similarnon-mass spectrometry based technique such that base count data isgenerated; and (f) querying a database comprising at least one basecount entry corresponding to the target nucleic acid to produce a matchof the base count data with said base count entry, thereby identifyingthe target nucleic acid. In certain embodiments, the methods furthercomprise a step after step (a) but before step (b) of treating thesample comprising amplified sequences such that either the sense strandscorresponding to the forward primer or said anti-sense strandscorresponding to the reverse primer of the amplified sequences areremoved from the sample in order to generate a purified sample. Incertain embodiments, step (c) employs a phosphodiesterase (e.g., such assnake venom phosphodiestease I).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary and detailed description is better understood whenread in conjunction with the accompanying drawings which are included byway of example and not by way of limitation.

FIG. 1 shows one embodiment of a bioagent identification process of thepresent invention.

FIG. 2 shows one embodiment of a bioagent identification process of thepresent invention.

FIG. 3 shows one embodiment of a sequencing system composed of zero-movewaveguides.

FIG. 4 shows a process diagram illustrating one embodiment of the primerpair selection process.

FIG. 5 shows a process diagram illustrating one embodiment of the primerpair validation process. Here select primers are shown meeting testcriteria. Criteria include but are not limited to, the ability toamplify targeted bioagent nucleic acid, the ability to excludenon-target bioagents, the ability to not produce unexpected amplicons,the ability to not dimerize, the ability to have analytical limits ofdetection of ≦100 genomic copies/reaction, and the ability todifferentiate amongst different target organisms.

FIG. 6 shows a block diagram showing a representative system.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention provides systems, methods, and compositions fornucleic acid detection based on non-mass determined base compositions.For example, in certain embodiments, base count data for a templatenucleic acid is generated without measuring the molecular mass of thetemplate nucleic acid (e.g., by sequencing the template nucleic acid)and a database comprising base count entries is queried to identify thetarget nucleic acid. In particular embodiments, sequencing is employedwhich is conducted in substantially real-time.

The present invention provides methods that are useful in detecting andcharacterizing nucleic acids. The methods typically involve determiningat least partial base counts (i.e., partial or complete base counts)and/or the sequence of template or target nucleic acids in real-timewithout measuring the molecular mass of the template nucleic acid.Exemplary applications of the method include diagnosing infectious orgenetic diseases, forensics, genotyping, and companion diagnostics,among many other applications. In addition, the invention also providesrelated zero-mode waveguide (e.g., arrays of zero-mode waveguides),kits, computer program products and systems.

The term “partial base composition” or “partial base count” refers tothe number of each residue of at least one nucleobase type (e.g., agiven purine nucleobase type, a given pyrimidine nucleobase type, agiven nucleobase analog type, and/or the like), but not each residuecomprised in an amplicon or other nucleic acid (e.g., for single ormultiple strands of those nucleic acids), without consideration for thelinear arrangement of these residues in the strand(s) of the amplicon.For example, if a given amplicon or other nucleic acid includes fournucleobase types (e.g., adenosine (A), guanosine (G), cytidine, (C), and(deoxy)thymidine (T)), a partial base count for that amplicon or othernucleic acid would include the number of any one of those fournucleobase types (e.g., [A_(w)], [G_(x)], [C_(y)], or [T_(x)]), any twoof those four nucleobase types (e.g., [A_(w)G_(x)], [A_(w)C_(y)],[A_(w)T_(z)], [G_(x)C_(y)], [G_(x)T_(z)], or [C_(y)T_(z)]), or at mostany three of those four nucleobase types (e.g., [A_(w)G_(x)C_(y)],[A_(w)C_(y)T_(z)], [A_(w)G_(x)T_(z)], or [G_(x)C_(y)T_(z)]), in which w,x, y and z are each independently a whole number representing the numberof said nucleoside residues in that amplicon or other nucleic acid. Tofurther illustrate, if a nucleic acid has the following composition:ATTGCCTAAGGTTAACG, then partial base counts for that nucleic acidinclude [A₅], [G₄], [C₃], [T₅], [A₅G₄], [A₅C₃], [A₅T₅], [G₄C₃], [G₄T₅],[C₃T₅], [A₅G₄C₃], [A₅C₃T₅], [A₅G₄T₅], or [G₄C₃T₅].

FIG. 1 schematically illustrates an exemplary process according to oneembodiment of the invention. As shown, the process includes obtaining asample with template nucleic acids. The sample is prepared, for example,by purifying or at partially isolating the template nucleic acids. Asshown, the template nucleic acids are amplified, for example, inpolymerase chain reactions or other amplification approaches to produceamplicons. In some embodiments, template nucleic acids are analyzeddirectly, for example, without being amplified. Amplicon base countsand/or sequences are determined, for example, using an array ofzero-mode waveguides and a fluorescent microscope (and, e.g., primernucleic acids (e.g., intelligent primers), polymerases (e.g., Φ29 DNApolymerases, etc.), phospholinked nucleotides, etc, as also describedin, for example, Korlach et al. (2008) “Selective aluminum passivationfor targeted immobilization of single DNA polymerase molecules inzero-mode waveguide nanostructures” Proc. Nat'l. Acad. Sci. U.S.A.105(4):1176-1181, herein incorporated by reference. The determinedamplicon base counts and/or amplicon sequence information is used toquery a base counts and/or sequence database to detect, identify,characterize, etc. the template nucleic acids. See also, FIG. 2 foradditional exemplary variations.

Sequencing Technologies

As described above, embodiments of the present invention involvedetermining the base composition, or partial base composition, or atarget sequence without determining the mass of the target sequence(e.g., without using mass spectrometry related methods). In certainembodiments, such methods of determining base compositions employsequencing methods. The present invention is not limited by the type ofsequencing method employed. Exemplary sequencing methods are describedbelow.

Illustrative non-limiting examples of nucleic acid sequencing techniquesinclude, but are not limited to, chain terminator (Sanger) sequencingand dye terminator sequencing. Those of ordinary skill in the art willrecognize that because RNA is less stable in the cell and more prone tonuclease attack experimentally RNA is usually reverse transcribed to DNAbefore sequencing.

Chain terminator sequencing uses sequence-specific termination of a DNAsynthesis reaction using modified nucleotide substrates. Extension isinitiated at a specific site on the template DNA by using a shortradioactive, or other labeled, oligonucleotide primer complementary tothe template at that region. The oligonucleotide primer is extendedusing a DNA polymerase, standard four deoxynucleotide bases, and a lowconcentration of one chain terminating nucleotide, most commonly adi-deoxynucleotide. This reaction is repeated in four separate tubeswith each of the bases taking turns as the di-deoxynucleotide. Limitedincorporation of the chain terminating nucleotide by the DNA polymeraseresults in a series of related DNA fragments that are terminated only atpositions where that particular di-deoxynucleotide is used. For eachreaction tube, the fragments are size-separated by electrophoresis in aslab polyacrylamide gel or a capillary tube filled with a viscouspolymer. The sequence is determined by reading which lane produces avisualized mark from the labeled primer as you scan from the top of thegel to the bottom.

Dye terminator sequencing alternatively labels the terminators. Completesequencing can be performed in a single reaction by labeling each of thedi-deoxynucleotide chain-terminators with a separate fluorescent dye,which fluoresces at a different wavelength.

A set of methods referred to as “next-generation sequencing” techniqueshave emerged as alternatives to Sanger and dye-terminator sequencingmethods (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLeanet al., Nature Rev. Microbiol., 7: 287-296; each herein incorporated byreference in their entirety). Next-generation sequencing (NGS) methodsshare the common feature of massively parallel, high-throughputstrategies, with the goal of lower costs in comparison to oldersequencing methods. NGS methods can be broadly divided into those thatrequire template amplification and those that do not.Amplification-requiring methods include pyrosequencing commercialized byRoche as the 454 technology platforms (e.g., GS 20 and GS FLX), theSolexa platform commercialized by Illumina, and the SupportedOligonucleotide Ligation and Detection (SOLiD) platform commercializedby Applied Biosystems. Non-amplification approaches, also known assingle-molecule sequencing, are exemplified by the HeliScope platformcommercialized by Helicos BioSciences, and emerging platformscommercialized by VisiGen, Oxford Nanopore Technologies Ltd., andPacific Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658,2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No.6,210,891; U.S. Pat. No. 6,258,568; each herein incorporated byreference in its entirety), template DNA is fragmented, end-repaired,ligated to adaptors, and clonally amplified in-situ by capturing singletemplate molecules with beads bearing oligonucleotides complementary tothe adaptors. Each bead bearing a single template type iscompartmentalized into a water-in-oil microvesicle, and the template isclonally amplified using a technique referred to as emulsion PCR. Theemulsion is disrupted after amplification and beads are deposited intoindividual wells of a picotitre plate functioning as a flow cell duringthe sequencing reactions. Ordered, iterative introduction of each of thefour dNTP reagents occurs in the flow cell in the presence of sequencingenzymes and luminescent reporter such as luciferase. In the event thatan appropriate dNTP is added to the 3′ end of the sequencing primer, theresulting production of ATP causes a burst of luminescence within thewell, which is recorded using a CCD camera. It is possible to achieveread lengths greater than or equal to 400 bases, and 1×10⁶ sequencereads can be achieved, resulting in up to 500 million base pairs (Mb) ofsequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S.Pat. No. 6,833,246; U.S. Pat. No. 7,115,400; U.S. Pat. No. 6,969,488;each herein incorporated by reference in its entirety), sequencing dataare produced in the form of shorter-length reads. In this method,single-stranded fragmented DNA is end-repaired to generate5′-phosphorylated blunt ends, followed by Klenow-mediated addition of asingle A base to the 3′ end of the fragments. A-addition facilitatesaddition of T-overhang adaptor oligonucleotides, which are subsequentlyused to capture the template-adaptor molecules on the surface of a flowcell that is studded with oligonucleotide anchors. The anchor is used asa PCR primer, but because of the length of the template and itsproximity to other nearby anchor oligonucleotides, extension by PCRresults in the “arching over” of the molecule to hybridize with anadjacent anchor oligonucleotide to form a bridge structure on thesurface of the flow cell. These loops of DNA are denatured and cleaved.Forward strands are then sequenced with reversible dye terminators. Thesequence of incorporated nucleotides is determined by detection ofpost-incorporation fluorescence, with each fluor and block removed priorto the next cycle of dNTP addition. Sequence read length ranges from 36nucleotides to over 50 nucleotides, with overall output exceeding 1billion nucleotide pairs per analytical run.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding etal., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148; U.S. Pat. No.6,130,073; each herein incorporated by reference in their entirety) alsoinvolves fragmentation of the template, ligation to oligonucleotideadaptors, attachment to beads, and clonal amplification by emulsion PCR.Following this, beads bearing template are immobilized on a derivatizedsurface of a glass flow-cell, and a primer complementary to the adaptoroligonucleotide is annealed. However, rather than utilizing this primerfor 3′ extension, it is instead used to provide a 5′ phosphate group forligation to interrogation probes containing two probe-specific basesfollowed by 6 degenerate bases and one of four fluorescent labels. Inthe SOLiD system, interrogation probes have 16 possible combinations ofthe two bases at the 3′ end of each probe, and one of four fluors at the5′ end. Fluor color and thus identity of each probe corresponds tospecified color-space coding schemes. Multiple rounds (usually 7) ofprobe annealing, ligation, and fluor detection are followed bydenaturation, and then a second round of sequencing using a primer thatis offset by one base relative to the initial primer. In this manner,the template sequence can be computationally re-constructed, andtemplate bases are interrogated twice, resulting in increased accuracy.Sequence read length averages 35 nucleotides, and overall output exceeds4 billion bases per sequencing run.

In certain embodiments, nanopore sequencing in employed (see, e.g.,Astier et al., J Am Chem Soc. 2006 Feb. 8; 128(5):1705-10, hereinincorporated by reference). The theory behind nanopore sequencing has todo with what occurs when the nanopore is immersed in a conducting fluidand a potential (voltage) is applied across it: under these conditions aslight electric current due to conduction of ions through the nanoporecan be observed, and the amount of current is exceedingly sensitive tothe size of the nanopore. If DNA molecules pass (or part of the DNAmolecule passes) through the nanopore, this can create a change in themagnitude of the current through the nanopore, thereby allowing thesequences of the DNA molecule to be determined.

HeliScope by Helicos BioSciences (Voelkerding et al., Clinical Chem.,55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296;U.S. Pat. No. 7,169,560; U.S. Pat. No. 7,282,337; U.S. Pat. No.7,482,120; U.S. Pat. No. 7,501,245; U.S. Pat. No. 6,818,395; U.S. Pat.No. 6,911,345; U.S. Pat. No. 7,501,245; each herein incorporated byreference in their entirety) is the first commercialized single-moleculesequencing platform. This method does not require clonal amplification.Template DNA is fragmented and polyadenylated at the 3′ end, with thefinal adenosine bearing a fluorescent label. Denatured polyadenylatedtemplate fragments are ligated to poly(dT) oligonucleotides on thesurface of a flow cell. Initial physical locations of captured templatemolecules are recorded by a CCD camera, and then label is cleaved andwashed away. Sequencing is achieved by addition of polymerase and serialaddition of fluorescently-labeled dNTP reagents. Incorporation eventsresult in fluor signal corresponding to the dNTP, and signal is capturedby a CCD camera before each round of dNTP addition. Sequence read lengthranges from 25-50 nucleotides, with overall output exceeding 1 billionnucleotide pairs per analytical run.

Another exemplary nucleic acid sequencing approach developed by StratosGenomics, Inc. that is also optionally adapted for use with the presentinvention involves the use of Xpandomers. This sequencing processtypically includes providing a daughter strand produced by atemplate-directed synthesis. The daughter strand generally includes aplurality of subunits coupled in a sequence corresponding to acontiguous nucleotide sequence of all or a portion of a target nucleicacid in which the individual subunits comprise a tether, at least oneprobe or nucleobase residue, and at least one selectively cleavablebond. The selectively cleavable bond(s) is/are cleaved to yield anXpandomer of a length longer than the plurality of the subunits of thedaughter strand. The Xpandomer typically includes the tethers andreporter elements for parsing genetic information in a sequencecorresponding to the contiguous nucleotide sequence of all or a portionof the target nucleic acid. Reporter elements of the Xpandomer are thendetected. Additional details relating to Xpandomer-based approaches aredescribed in, for example, U.S. Patent Publication No. 20090035777,entitled “HIGH THROUGHPUT NUCLEIC ACID SEQUENCING BY EXPANSION,” thatwas filed Jun. 19, 2008, which is incorporated herein in its entirety.

Other emerging single molecule sequencing methods include real-timesequencing by synthesis using a VisiGen platform (Voelkerding et al.,Clinical Chem., 55: 641-658, 2009; U.S. Pat. No. 7,329,492; U.S. patentapplication Ser. No. 11/671,956; U.S. patent application Ser. No.11/781,166; each herein incorporated by reference in their entirety) inwhich immobilized, primed DNA template is subjected to strand extensionusing a fluorescently-modified polymerase and florescent acceptormolecules, resulting in detectible fluorescence resonance energytransfer (FRET) upon nucleotide addition.

Another real-time single molecule sequencing system developed by PacificBiosciences (Voelkerding et al., Clinical Chem., 55: 641-658, 2009;MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No.7,170,050; U.S. Pat. No. 7,302,146; U.S. Pat. No. 7,313,308; U.S. Pat.No. 7,476,503; all of which are herein incorporated by reference)utilizes reaction wells 50-100 nm in diameter and encompassing areaction volume of approximately 20 zeptoliters (10×10⁻²¹ L). Sequencingreactions are performed using immobilized template, modified phi29 DNApolymerase, and high local concentrations of fluorescently labeleddNTPs. High local concentrations and continuous reaction conditionsallow incorporation events to be captured in real time by fluor signaldetection using laser excitation, an optical waveguide, and a CCDcamera.

In certain embodiments, the single molecule real time (SMRT) DNAsequencing methods using zero-mode waveguides (ZMWs) developed byPacific Biosciences, or similar methods, are employed. With thistechnology, DNA sequencing is performed on SMRT chips, each containingthousands of zero-mode waveguides (ZMWs). A ZMW is a hole, tens ofnanometers in diameter, fabricated in a 100 nm metal film deposited on asilicon dioxide substrate. Each ZMW becomes a nanophotonic visualizationchamber providing a detection volume of just 20 zeptoliters (10-21liters). At this volume, the activity of a single molecule can bedetected amongst a background of thousands of labeled nucleotides.

The ZMW provides a window for watching DNA polymerase as it performssequencing by synthesis. Within each chamber, a single DNA polymerasemolecule is attached to the bottom surface such that it permanentlyresides within the detection volume. Phospholinked nucleotides, eachtype labeled with a different colored fluorophore, are then introducedinto the reaction solution at high concentrations which promote enzymespeed, accuracy, and processivity. Due to the small size of the ZMW,even at these high, biologically relevant concentrations, the detectionvolume is occupied by nucleotides only a small fraction of the time. Inaddition, visits to the detection volume are fast, lasting only a fewmicroseconds, due to the very small distance that diffusion has to carrythe nucleotides. The result is a very low background.

As the DNA polymerase incorporates complementary nucleotides, each baseis held within the detection volume for tens of milliseconds, which isorders of magnitude longer than the amount of time it takes a nucleotideto diffuse in and out of the detection volume. During this time, theengaged fluorophore emits fluorescent light whose color corresponds tothe base identity. Then, as part of the natural incorporation cycle, thepolymerase cleaves the bond holding the fluorophore in place and the dyediffuses out of the detection volume. Following incorporation, thesignal immediately returns to baseline and the process repeats.

Unhampered and uninterrupted, the DNA polymerase continues incorporatingbases at a speed of tens per second. In this way, a completely naturallong chain of DNA is produced in minutes. Simultaneous and continuousdetection occurs across all of the thousands of ZMWs on the SMRT chip inreal time. This approach has the capability to produce reads thousandsof nucleotides in length.

Some aspects the invention are generally directed to optical detectionor monitoring systems (e.g., ZMVs discussed above), methods enabled bysuch systems, and components of such systems for monitoring, inreal-time, optical signals that emanate from multiple discrete sourcesof those optical signals. In particular, the optical detection andmonitoring systems are generally capable of monitoring discrete signalsfrom potentially very large numbers of different signal sources,optionally separating and/or deconvolving such signals into constituentsignal events, and doing so in real-time, despite that such signals maybe changing rapidly, over time.

The systems typically include all or a portion of a collection ofdifferent functional elements. These elements include the multiplediscrete sources that include the capability of generating opticalsignals. In some aspects, such sources include chemical, biochemicaland/or biological reactants, or mimics of such reactants that arecapable of generating optical signals that are indicative of theirpresence, reaction or conversion. While the sources may be capable ofgenerating optical signals on their own, in certain cases, a source ofexcitation radiation is also provided to excite optical signals, e.g.,fluorescence, within the sources.

The systems also typically include optical elements that direct,separate, and/or otherwise alter optical signals from these sources (aswell as excitation radiation directed at such sources), in order toultimately derive optimal amounts of information from such signals whenthey are ultimately detected. Consequently, the systems typicallyinclude an optical detection system for detecting the potentially largenumbers of signals that were directed from the sources, and optionallyseparated and/or otherwise altered by the optical elements. Signalsdetected by the optical detection system are then recorded and processedby appropriate processing systems and data management processes toprovide output of the system in user ready formats.

The systems are typically applied in the monitoring of arrays orcollections of spatially discrete chemical, biochemical and/orbiological reactions that generate optically detectable signals, such aschromogenic reactions, luminescent or luminogenic reactions, orfluorescent or fluorogenic reactions. A few examples of reactionsinclude those that are regularly performed in the pharmaceutical,biotechnology and healthcare diagnostic fields, i.e., base countanalyses, immunoassays, enzymatic assays, receptor assays, nucleic acidhybridization assays, nucleic acid synthesis reactions, cellular assays,and many others.

Typically, the progress of the reactions used in application of thesystems described herein result in one or more of the consumption,production and/or conversion of a material that is capable of generatingan optically detectable signal, either alone, or in response to anexternal stimulus, e.g., excitation radiation. By way of example,certain reactants may become fluorescent upon reaction with anotherreactant, or may have their fluorescence altered or reduced upon suchreaction. As such, the fluorescence emitted from the reaction inresponse to an excitation radiation will change as the reactionprogresses. The systems generally provide for the source of suchsignals, e.g., the area in which the reaction occurs, includingoptionally, the reactants and/or products, the optical elements forcollecting, directing and optionally separating and/or altering suchsignals from such sources, and the ultimate detection of such signals,as well as the manipulation of the resulting data to yield optimal valueand information for the user.

The systems typically include all or a subset of a substrate thatincludes all or a subset of the sources of optical signals, an optionalexcitation light source, an optical train that includes the variousoptical elements for collection, direction and/or manipulation of theoptical signals and optional excitation light, optical detectors forreceiving, detecting and recording (or putting into a form forrecordation) the optical signals, as well as processors for processingdata derived from the optical detectors.

A general schematic representation of the system as set forth above, isillustrated in FIG. 3. As shown, the system 100 includes a substrate 102that includes a plurality of discrete sources of optical signals, e.g.,reaction wells or optical confinements 104. An excitation light source,e.g., laser 106, is optionally provided in the system and is positionedto direct excitation radiation at the various signal sources. This istypically done by directing excitation radiation at or throughappropriate optical components, e.g., dichroic 108 and objective lens110, that direct the excitation radiation at the substrate 102, andparticularly the signal sources 104. Emitted signals from source 104 arethen collected by the optical components, e.g., objective 110, andpassed through additional optical elements, e.g., dichroic 108, prism 12and lens 114, until they are directed to and impinge upon an opticaldetection system, e.g., detector array 116. The signals are thendetected by detector array 116, and the data from that detection istransmitted to an appropriate data processing unit, e.g., computer 118,where the data is subjected to interpretation, analysis, and ultimatelypresented in a user ready format, e.g., on display 120, or printout 122,from printer 124.

Processes and systems for such real time sequencing that may be adaptedfor use with the invention are described in, for example, U.S. Pat. No.7,405,281, entitled “Fluorescent nucleotide analogs and uses therefor”,issued Jul. 29, 2008 to Xu et al., U.S. Pat. No. 7,315,019, entitled“Arrays of” optical confinements and uses thereof, issued Jan. 1, 2008to Turner et al., U.S. Pat. No. 7,313,308, entitled “Optical analysis ofmolecules”, issued Dec. 25, 2007 to Turner et al., U.S. Pat. No.7,302,146, entitled “Apparatus and method for analysis of molecules”,issued Nov. 27, 2007 to Turner et al., and U.S. Pat. No. 7,170,050,entitled “Apparatus and methods for optical analysis of molecules”,issued Jan. 30, 2007 to Turner et al., U.S. Patent Publications Nos.20080212960, entitled “Methods and systems for simultaneous real-timemonitoring of optical signals from multiple sources”, filed Oct. 26,2007 by Lundquist et al., 20080206764, entitled “Flowcell system forsingle molecule detection”, filed Oct. 26, 2007 by Williams et al.,20080199932, entitled “Active surface coupled polymerases”, filed Oct.26, 2007 by Hanzel et al., 20080199874, entitled “CONTROLLABLE STRANDSCISSION OF MINI CIRCLE DNA”, filed Feb. 11, 2008 by Otto et al.,20080176769, entitled “Articles having localized molecules disposedthereon and methods of producing same”, filed Oct. 26, 2007 by Rank etal., 20080176316, entitled “Mitigation of photodamage in analyticalreactions”, filed Oct. 31, 2007 by Eid et al., 20080176241, entitled“Mitigation of photodamage in analytical reactions”, filed Oct. 31, 2007by Eid et al., 20080165346, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Oct. 26, 2007 by Lundquist et al., 20080160531, entitled“Uniform surfaces for hybrid material substrates and methods for makingand using same”, filed Oct. 31, 2007 by Korlach, 20080157005, entitled“Methods and systems for simultaneous real-time monitoring of opticalsignals from multiple sources”, filed Oct. 26, 2007 by Lundquist et al.,20080153100, entitled “Articles having localized molecules disposedthereon and methods of producing same”, filed Oct. 31, 2007 by Rank etal., 20080153095, entitled “CHARGE SWITCH NUCLEOTIDES”, filed Oct. 26,2007 by Williams et al., 20080152281, entitled “Substrates, systems andmethods for analyzing materials”, filed Oct. 31, 2007 by Lundquist etal., 20080152280, entitled “Substrates, systems and methods foranalyzing materials”, filed Oct. 31, 2007 by Lundquist et al.,20080145278, entitled “Uniform surfaces for hybrid material substratesand methods for making and using same”, filed Oct. 31, 2007 by Korlach,20080128627, entitled “SUBSTRATES, SYSTEMS AND METHODS FOR ANALYZINGMATERIALS”, filed Aug. 31, 2007 by Lundquist et al., 20080108082,entitled “Polymerase enzymes and reagents for enhanced nucleic acidsequencing”, filed Oct. 22, 2007 by Rank et al., 20080095488, entitled“SUBSTRATES FOR PERFORMING ANALYTICAL REACTIONS”, filed Jun. 11, 2007 byFoquet et al., 20080080059, entitled “MODULAR OPTICAL COMPONENTS ANDSYSTEMS INCORPORATING SAME”, filed Sep. 27, 2007 by Dixon et al.,20080050747, entitled “Articles having localized molecules disposedthereon and methods of producing and using same”, filed Aug. 14, 2007 byKorlach et al., 20080032301, entitled “Articles having localizedmolecules disposed thereon and methods of producing same”, filed Mar.29, 2007 by Rank et al., 20080030628, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Feb. 9, 2007 by Lundquist et al., 20080009007, entitled“CONTROLLED INITIATION OF PRIMER EXTENSION”, filed Jun. 15, 2007 by Lyleet al., 20070238679, entitled “Articles having localized moleculesdisposed thereon and methods of producing same”, filed Mar. 30, 2006 byRank et al., 20070231804, entitled “Methods, systems and compositionsfor monitoring enzyme activity and applications thereof”, filed Mar. 31,2006 by Korlach et al., 20070206187, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Feb. 9, 2007 by Lundquist et al., 20070196846, entitled“Polymerases for nucleotide analogue incorporation”, filed Dec. 21, 2006by Hanzel et al., 20070188750, entitled “Methods and systems forsimultaneous real-time monitoring of optical signals from multiplesources”, filed Jul. 7, 2006 by Lundquist et al., 20070161017, entitled“MITIGATION OF PHOTODAMAGE IN ANALYTICAL REACTIONS”, filed Dec. 1, 2006by Eid et al., 20070141598, entitled “Nucleotide Compositions and UsesThereof”, filed Nov. 3, 2006 by Turner et al., 20070134128, entitled“Uniform surfaces for hybrid material substrate and methods for makingand using same”, filed Nov. 27, 2006 by Korlach, 20070128133, entitled“Mitigation of photodamage in analytical reactions”, filed Dec. 2, 2005by Eid et al., 20070077564, entitled “Reactive surfaces, substrates andmethods of producing same”, filed Sep. 30, 2005 by Roitman et al.,20070072196, entitled “Fluorescent nucleotide analogs and usestherefore”, filed Sep. 29, 2005 by Xu et al., and 20070036511, entitled“Methods and systems for monitoring multiple optical signals from asingle source”, filed Aug. 11, 2005 by Lundquist et al., and Korlach etal. (2008) “Selective aluminum passivation for targeted immobilizationof single DNA polymerase molecules in zero-mode waveguidenanostructures” Proc. Nat'l. Acad. Sci. U.S.A. 105(4): 11761181—all ofwhich are herein incorporated by reference in their entireties.

DEFINITIONS AND FURTHER DESCRIPTION

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. Further, unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. In describing and claiming the present invention, thefollowing terminology and grammatical variants will be used inaccordance with the definitions set forth below.

As used herein, the term “about” means encompassing plus or minus 10%.For example, about 200 nucleotides refers to a range encompassingbetween 180 and 220 nucleotides.

As used herein, the term “amplicon” or “bioagent identifying amplicon”refers to a nucleic acid generated using the primer pairs describedherein. The amplicon is typically double stranded DNA; however, it maybe RNA and/or DNA:RNA. In some embodiments, the amplicon comprises DNAcomplementary to HPV RNA, DNA, or cDNA. In some embodiments, theamplicon comprises sequences of conserved regions/primer pairs andintervening variable region. As discussed herein, primer pairs areconfigured to generate amplicons from bioagent nucleic acid. As such,the base composition of any given amplicon may include the primer pair,the complement of the primer pair, the conserved regions and thevariable region from the bioagent that was amplified to generate theamplicon. One skilled in the art understands that the incorporation ofthe designed primer pair sequences into an amplicon may replace thenative sequences at the primer binding site, and complement thereof. Incertain embodiments, after amplification of the target region using theprimers the resultant amplicons having the primer sequences are used togenerate the base composition data. Bioagent identifying ampliconsgenerate base compositions that are preferably unique to the identity ofa bioagent.

The term “amplifying” or “amplification” in the context of nucleic acidsrefers to the production of multiple copies of a polynucleotide, or aportion of the polynucleotide, typically starting from a small amount ofthe polynucleotide (e.g., a single polynucleotide molecule), where theamplification products or amplicons are generally detectable.Amplification of polynucleotides encompasses a variety of chemical andenzymatic processes. The generation of multiple DNA copies from one or afew copies of a target or template DNA molecule during a polymerasechain reaction (PCR) or a ligase chain reaction (LCR) are forms ofamplification. Amplification is not limited to the strict duplication ofthe starting molecule. For example, the generation of multiple cDNAmolecules from a limited amount of RNA in a sample using reversetranscription (RT)-PCR is a form of amplification. Furthermore, thegeneration of multiple RNA molecules from a single DNA molecule duringthe process of transcription is also a form of amplification.

The term “attached” refers to interactions including, but not limitedto, covalent bonding, ionic bonding, chemisorption, physisorption, andcombinations thereof.

As used herein, “viral nucleic acid” includes, but is not limited to,DNA, RNA, or DNA that has been obtained from viral RNA, such as, forexample, by performing a reverse transcription reaction. Viral RNA caneither be single-stranded (of positive or negative polarity) ordouble-stranded.

As used herein, the term “base composition” or “base count” refers tothe number of each residue comprised in an amplicon or other nucleicacid, without consideration for the linear arrangement of these residuesin the strand(s) of the amplicon. The amplicon residues comprise,adenosine (A), guanosine (G), cytidine, (C), (deoxy)thymidine (T),uracil (U), inosine (I), nitroindoles such as 5-nitroindole or3-nitropyrrole, dP or dK (Hill F et al. Polymerase recognition ofsynthetic oligodeoxyribonucleotides incorporating degenerate pyrimidineand purine bases. Proc Natl Acad Sci USA. 1998 Apr. 14; 95(8):4258-63),an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot etal., Nucleosides and Nucleotides, 1995, 14, 1053-1056), the purineanalog 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide,2,6-diaminopurine, 5-propynyluracil, 5-propynylcytosine, phenoxazines,including G-clamp, 5-propynyl deoxy-cytidine, deoxy-thymidinenucleotides, 5-propynylcytidine, 5-propynyluridine and mass tag modifiedversions thereof, including 7-deaza-2′-deoxyadenosine-5-triphosphate,5-iodo-2′-deoxyuridine-5′-triphosphate,5-bromo-2′-deoxyuridine-5′-triphosphate,5-bromo-2′-deoxycytidine-5′-triphosphate,5-iodo-2′-deoxycytidine-5′-triphosphate,5-hydroxy-2′-deoxyuridine-5′-triphosphate,4-thiothymidine-5′-triphosphate, 5-aza-2′-deoxyuridine-5′-triphosphate,5-fluoro-2′-deoxyuridine-5′-triphosphate,O6-methyl-2′-deoxyguanosine-5′-triphosphate,N2-methyl-2′-deoxyguanosine-5′-triphosphate,8-oxo-2′-deoxyguanosine-5′-triphosphate orthiothymidine-5′-triphosphate. In some embodiments, the mass-modifiednucleobase comprises ¹⁵N or ¹³C or both ¹⁵N and ¹³C. In someembodiments, the non-natural nucleosides used herein include5-propynyluracil, 5-propynylcytosine and inosine. Herein the basecomposition for an unmodified DNA amplicon is notated asA_(w)G_(x)C_(y)T_(z), wherein w, x, y and z are each independently awhole number representing the number of said nucleoside residues in anamplicon. Base compositions for amplicons comprising modifiednucleosides are similarly notated to indicate the number of said naturaland modified nucleosides in an amplicon.

As used herein, a “base composition probability cloud” is arepresentation of the diversity in base composition resulting from avariation in sequence that occurs among different isolates of a givenspecies, family or genus. Base composition calculations for a pluralityof amplicons are mapped on a pseudo four-dimensional plot. Relatedmembers in a family, genus or species typically cluster within thisplot, forming a base composition probability cloud.

As used herein, the term “base composition signature” refers to the basecomposition generated by any one particular amplicon.

As used herein, a “bioagent” means any biological organism or componentthereof or a sample containing a biological organism or componentthereof, including microorganisms or infectious substances, or anynaturally occurring, bioengineered or synthesized component of any suchmicroorganism or infectious substance or any nucleic acid derived fromany such microorganism or infectious substance. Those of ordinary skillin the art will understand fully what is meant by the term bioagentgiven the instant disclosure. Still, a non-exhaustive list of bioagentsincludes: cells, cell lines, human clinical samples, mammalian bloodsamples, cell cultures, bacterial cells, viruses, viroids, fungi,protists, parasites, rickettsiae, protozoa, animals, mammals or humans.Samples may be alive, non-replicating or dead or in a vegetative state(for example, vegetative bacteria or spores).

As used herein, a “bioagent division” is defined as group of bioagentsabove the species level and includes but is not limited to, orders,families, genus, classes, clades, genera or other such groupings ofbioagents above the species level.

As used herein, “broad range survey primers” are primers designed toidentify an unknown bioagent as a member of a particular biologicaldivision (e.g., an order, family, class, Glade, or genus). However, insome cases the broad range survey primers are also able to identifyunknown bioagents at the species or sub-species level. As used herein,“division-wide primers” are primers designed to identify a bioagent atthe species level and “drill-down” primers are primers designed toidentify a bioagent at the sub-species level. As used herein, the“sub-species” level of identification includes, but is not limited to,strains, subtypes, variants, and isolates. Drill-down primers are notalways required for identification at the sub-species level becausebroad range survey intelligent primers may, in some cases providesufficient identification resolution to accomplishing thisidentification objective. Broad range survey primers may be used in thenon-mass determined base compositions methods and systems of the presentinvention.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence“5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids.

The term “conserved region” in the context of nucleic acids refers to anucleobase sequence (e.g., a subsequence of a nucleic acid, etc.) thatis the same or similar in two or more different regions or segments of agiven nucleic acid molecule (e.g., an intramolecular conserved region),or that is the same or similar in two or more different nucleic acidmolecules (e.g., an intermolecular conserved region). To illustrate, aconserved region may be present in two or more different taxonomic ranks(e.g., two or more different genera, two or more different species, twoor more different subspecies, and the like) or in two or more differentnucleic acid molecules from the same organism. To further illustrate, incertain embodiments, nucleic acids comprising at least one conservedregion typically have between about 70%-100%, between about 80-100%,between about 90-100%, between about 95-100%, or between about 99-100%sequence identity in that conserved region. A conserved region may alsobe selected or identified functionally as a region that permitsgeneration of amplicons via primer extension through hybridization of acompletely or partially complementary primer to the conserved region foreach of the target sequences to which conserved region is conserved.

As used herein, in some embodiments the term “database” is used to referto a collection of base composition and/or partial base compositiondata. The base composition data in the database is indexed to bioagentsand to primer pairs. The base composition data reported in the databasecomprises the number of at least one type of nucleoside in an amplicon(e.g., A₁₇) that would be generated for each bioagent using each primer.The database can be populated by empirical data. In this aspect ofpopulating the database, a bioagent is selected and a primer pair isused to generate an amplicon. Note that base composition entries in thedatabase may be derived from sequencing data (i.e., known sequenceinformation). An entry in the database is made to associate correlatethe base composition with the bioagent and the primer pair used. Thedatabase may also be populated using other databases comprising bioagentinformation. For example, using the GenBank database it is possible toperform electronic PCR using an electronic representation of a primerpair. This in silico method may provide the base composition for any orall selected bioagent(s) stored in the GenBank database. The informationmay then be used to populate the base composition database as describedabove. A base composition database can be in silico, a written table, areference book, a spreadsheet or any form generally amenable todatabases. Preferably, it is in silico on computer readable media.

The term “detect”, “detecting” or “detection” refers to an act ofdetermining the existence or presence of one or more targets (e.g.,bioagent nucleic acids, amplicons, etc.) in a sample.

As used herein, the term “etiology” refers to the causes or origins, ofdiseases or abnormal physiological conditions.

Nucleic acids are “extended” or “elongated” when additional nucleotides(or other analogous molecules) are incorporated into the nucleic acids.For example, a nucleic acid is optionally extended by a nucleotideincorporating biocatalyst, such as a polymerase that typically addsnucleotides at the 3′ terminal end of a nucleic acid.

An “extended primer nucleic acid” refers to a primer nucleic acid towhich one or more additional nucleotides have been added or otherwiseincorporated (e.g., covalently bonded to).

As used herein, the term “gene” refers to a nucleic acid (e.g., DNA)sequence that comprises coding sequences necessary for the production ofa polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length sequence or fragment thereofare retained.

A “genotype” refers to all or part of the genetic constitution of anucleic acid molecule, cell or subject, or group of nucleic acidmolecules, cells or subjects. For example, a genotype includes theparticular mutations and/or alleles (e.g., polymorphisms, such as singlenucleotide polymorphisms (SNPs) or the like) present at a given locus ordistributed in a genome.

A “heterocyclic ring” refers to a monocyclic or bicyclic ring that iseither saturated, unsaturated, or aromatic, and which comprises one ormore heteroatoms independently selected from nitrogen, oxygen andsulfur. A heterocyclic ring may be attached to the sugar moiety, oranalog thereof, of a nucleotide of the invention via any heteroatom orcarbon atom. Exemplary heterocyclic rings include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl,pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl,isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl,imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl,phthalazinyl, quinazolinyl, and the like.

A “homocyclic ring” refers to a saturated or unsaturated (but notaromatic) carbocyclic ring, such as cyclopropane, cyclobutane,cyclopentane, cyclohexane, cycloheptane, cyclohexene, and the like.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to nucleic acid sequences that are notfound naturally associated with the gene sequences in the chromosome orare associated with portions of the chromosome not found in nature(e.g., genes expressed in loci where the gene is not normallyexpressed).

The terms “homology,” “homologous” and “sequence identity” refer to adegree of identity. There may be partial homology or complete homology.A partially homologous sequence is one that is less than 100% identicalto another sequence. Determination of sequence identity is described inthe following example: a primer 20 nucleobases in length which isotherwise identical to another 20 nucleobase primer but having twonon-identical residues has 18 of 20 identical residues (18/20=0.9 or 90%sequence identity). In another example, a primer 15 nucleobases inlength having all residues identical to a 15 nucleobase segment of aprimer 20 nucleobases in length would have 15/20=0.75 or 75% sequenceidentity with the 20 nucleobase primer. In context of the presentinvention, sequence identity is meant to be properly determined when thequery sequence and the subject sequence are both described and alignedin the 5′ to 3′ direction. Sequence alignment algorithms such as BLAST,will return results in two different alignment orientations. In thePlus/Plus orientation, both the query sequence and the subject sequenceare aligned in the 5′ to 3′ direction. On the other hand, in thePlus/Minus orientation, the query sequence is in the 5′ to 3′ directionwhile the subject sequence is in the 3′ to 5′ direction. It should beunderstood that with respect to the primers of the present invention,sequence identity is properly determined when the alignment isdesignated as Plus/Plus. Sequence identity may also encompass alternateor “modified” nucleobases that perform in a functionally similar mannerto the regular nucleobases adenine, thymine, guanine and cytosine withrespect to hybridization and primer extension in amplificationreactions. In a non-limiting example, if the 5-propynyl pyrimidinespropyne C and/or propyne T replace one or more C or T residues in oneprimer which is otherwise identical to another primer in sequence andlength, the two primers will have 100% sequence identity with eachother. In another non-limiting example, Inosine (I) may be used as areplacement for G or T and effectively hybridize to C, A or U (uracil).Thus, if inosine replaces one or more C, A or U residues in one primerwhich is otherwise identical to another primer in sequence and length,the two primers will have 100% sequence identity with each other. Othersuch modified or universal bases may exist which would perform in afunctionally similar manner for hybridization and amplificationreactions and will be understood to fall within this definition ofsequence identity.

As used herein, “housekeeping gene” or “core viral gene” refers to agene encoding a protein or RNA involved in basic functions required forsurvival and reproduction of a bioagent. Housekeeping genes include, butare not limited to, genes encoding RNA or proteins involved intranslation, replication, recombination and repair, transcription,nucleotide metabolism, amino acid metabolism, lipid metabolism, energygeneration, uptake, secretion and the like.

As used herein, the term “hybridization” or “hybridize” is used inreference to the pairing of complementary nucleic acids. Hybridizationand the strength of hybridization (i.e., the strength of the associationbetween the nucleic acids) is influenced by such factors as the degreeof complementary between the nucleic acids, stringency of the conditionsinvolved, the melting temperature (T_(m)) of the formed hybrid, and theG:C ratio within the nucleic acids. A single molecule that containspairing of complementary nucleic acids within its structure is said tobe “self-hybridized.” An extensive guide to nucleic hybridization may befound in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, part I, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier (1993), which is incorporated by reference.

A “label” refers to a moiety attached (covalently or non-covalently), orcapable of being attached, to a molecule, which moiety provides or iscapable of providing information about the molecule (e.g., descriptive,identifying, etc. information about the molecule). Exemplary labelsinclude fluorescent labels, non-fluorescent labels, colorimetric labels,chemiluminescent labels, bioluminescent labels, radioactive labels,mass-modifying groups, antibodies, antigens, biotin, haptens, andenzymes (including, e.g., peroxidase, phosphatase, etc.).

The term “kit” is used in reference to a combination of articles thatfacilitate a process, method, assay, analysis or manipulation of asample. Kits can contain instructions describing how to use the kit(e.g., instructions describing the methods of the invention), zero-modewaveguides, primer nucleic acids, nucleotide incorporating biocatalysts,labeled nucleotides, chemical reagents, as well as any other components.

A “linker” or “spacer” refers to a chemical moiety that covalently ornon-covalently (e.g., ionically, etc.) attaches a compound orsubstituent group to, e.g., a solid support, another compound or group,or the like. For example, a linker optionally attaches a label (e.g., afluorescent dye, a radioisotope, etc.) to a nucleotide or the like.Linkers are typically bifunctional chemical moieties and in certainembodiments, they comprise cleavable attachments, which can be cleavedby, e.g., heat, an enzyme, a chemical agent, electromagnetic radiation,etc. to release materials or compounds from, e.g., a solid support,another compound, etc. A careful choice of linker allows cleavage to beperformed under appropriate conditions compatible with the stability ofthe compound and assay method. Generally a linker has no specificbiological activity other than to, e.g., join chemical species togetheror to preserve some minimum distance or other spatial relationshipbetween such species. However, the constituents of a linker may beselected to influence some property of the linked chemical species suchas three-dimensional conformation, net charge, hydrophobicity, etc.Additional description of linker molecules is provided in, e.g., Lyttleet al. (1996) Nucleic Acids Res. 24(14):2793, Shchepino et al. (2001)Nucleosides, Nucleotides, & Nucleic Acids 20:369, Doronina et al (2001)Nucleosides, Nucleotides, & Nucleic Acids 20:1007, Trawick et al. (2001)Bioconjugate Chem. 12:900, Olejnik et al. (1998) Methods in Enzymology291:135, Pljevaljcic et al. (2003) J. Am. Chem. Soc. 125(12):3486, Ward,et. al., U.S. Pat. No. 4,711,955, Stavrianopoulos, U.S. Pat. No.4,707,352, and Stavrianopoulos, U.S. Pat. No. 4,707,440, which are eachincorporated by reference.

A “mass modifying” group modifies the mass, typically measured in termsof molecular weight as daltons, of a molecule that comprises the group.For example, mass modifying groups that increase the discriminationbetween at least two nucleic acids with single base differences in sizeor sequence can be used to facilitate sequencing using, e.g., molecularweight determinations.

A “mixture” refers to a combination of two or more different components.A “reaction mixture” refers a mixture that comprises molecules that canparticipate in and/or facilitate a given reaction.

The term “molecular mass” refers to the mass of a compound (e.g., anucleic acid, etc.) as determined, for example, using mass spectrometry.

A “modified” enzyme refers to an enzyme comprising a monomer sequence inwhich at least one monomer of the sequence differs from a monomer in areference sequence, such as a native or wild-type form of the enzyme oranother modified form of the enzyme, e.g., when the two sequences arealigned for maximum identity. Exemplary modifications include monomerinsertions, deletions, and substitutions. The modified enzymes (i.e.,protein- or nucleic acid-based catalysts) of the invention have been orare optionally created by various diversity generating methods. Althoughessentially any method can be used to produce a modified enzyme, certainexemplary techniques include recombining (e.g., via recursiverecombination, synthetic recombination, or the like) two or more nucleicacids encoding one or more parental enzymes, or by mutating one or morenucleic acids that encode enzymes, e.g., using recursive ensemblemutagenesis, cassette mutagenesis, random mutagenesis, in vivomutagenesis, site directed mutagenesis, or the like. A nucleic acidencoding a parental enzyme typically includes a gene that, through themechanisms of transcription and translation, produces an amino acidsequence corresponding to a parental enzyme, e.g., a native form of theenzyme. Modified enzymes also include chimeric enzymes that haveidentifiable component sequences (e.g., structural and/or functionaldomains, etc.) derived from two or more parents. Also included withinthe definition of modified enzymes are those comprising chemicalmodifications (e.g., attached substituent groups, altered substituentgroups, etc.) relative to a reference sequence.

A “moiety” or “group” refers to one of the portions into whichsomething, such as a molecule, is divided (e.g., a functional group,substituent group, or the like). For example, a nucleotide typicallycomprises a basic group (e.g., adenine, thymine, cytosine, guanine,uracil, or an analog basic group), a sugar moiety (e.g., a moietycomprising a sugar ring or an analog thereof), and one or more phosphategroups.

As used herein, the term “primer” or “primer nucleic acid” refers to anoligonucleotide, whether occurring naturally as in a purifiedrestriction digest or produced synthetically, that is capable of actingas a point of initiation of synthesis when placed under conditions inwhich synthesis of a primer extension product that is complementary to anucleic acid strand is induced (e.g., in the presence of nucleotides andan inducing agent such as a biocatalyst (e.g., a DNA polymerase or thelike) and at a suitable temperature and pH). The primer is typicallysingle stranded for maximum efficiency in amplification, but mayalternatively be double stranded. If double stranded, the primer isgenerally first treated to separate its strands before being used toprepare extension products. In some embodiments, the primer is anoligodeoxyribonucleotide. The primer is sufficiently long to prime thesynthesis of extension products in the presence of the inducing agent.The exact lengths of the primers will depend on many factors, includingtemperature, source of primer and the use of the method.

As used herein, “intelligent primers” or “primers” or “primer pairs,” insome embodiments, are oligonucleotides that are designed to bind toconserved sequence regions of one or more bioagent nucleic acids togenerate bioagent identifying amplicons. In some embodiments, the boundprimers flank an intervening variable region between the conservedbinding sequences. Upon amplification, the primer pairs yield ampliconse.g., amplification products that provide base composition variabilitybetween the two or more bioagents. The variability of the basecompositions allows for the identification of one or more individualbioagents from, e.g., two or more bioagents based on the basecomposition distinctions. In some embodiments, the primer pairs are alsoconfigured to generate amplicons amenable to molecular mass analysis.Further, the sequences of the primer members of the primer pairs are notnecessarily fully complementary to the conserved region of the referencebioagent. For example, in some embodiments, the sequences are designedto be “best fit” amongst a plurality of bioagents at these conservedbinding sequences. Therefore, the primer members of the primer pairshave substantial complementarity with the conserved regions of thebioagents, including the reference bioagent.

In some embodiments of the invention, the oligonucleotide primer pairsdescribed herein can be purified. As used herein, “purifiedoligonucleotide primer pair,” “purified primer pair,” or “purified”means an oligonucleotide primer pair that is chemically-synthesized tohave a specific sequence and a specific number of linked nucleosides.This term is meant to explicitly exclude nucleotides that are generatedat random to yield a mixture of several compounds of the same lengtheach with randomly generated sequence. As used herein, the term“purified” or “to purify” refers to the removal of one or morecomponents (e.g., contaminants) from a sample.

The term “nucleic acid” or “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N⁶-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxyl-methyl)-uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N⁶-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N⁶-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

As used herein, the term “nucleobase” is synonymous with other terms inuse in the art including “nucleotide,” “deoxynucleotide,” “nucleotideresidue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” ordeoxynucleotide triphosphate (dNTP). As is used herein, a nucleobaseincludes natural and modified residues, as described herein.

A “nucleoside” refers to a nucleic acid component that comprises a baseor basic group (e.g., comprising at least one homocyclic ring, at leastone heterocyclic ring, at least one aryl group, and/or the like)covalently linked to a sugar moiety (e.g., a ribose sugar, etc.), aderivative of a sugar moiety, or a functional equivalent of a sugarmoiety (e.g., an analog, such as carbocyclic ring). For example, when anucleoside includes a sugar moiety, the base is typically linked to a1′-position of that sugar moiety. As described above, a base can benaturally occurring (e.g., a purine base, such as adenine (A) or guanine(G), a pyrimidine base, such as thymine (T), cytosine (C), or uracil(U)), or non-naturally occurring (e.g., a 7-deazapurine base, apyrazolo[3,4-d]pyrimidine base, a propynyl-dN base, etc.). Exemplarynucleosides include ribonucleosides, deoxyribonucleosides,dideoxyribonucleosides, carbocyclic nucleosides, etc.).

A “nucleotide” refers to an ester of a nucleoside, e.g., a phosphateester of a nucleoside. For example, a nucleotide can include 1, 2, 3, ormore phosphate groups covalently linked to a 5′ position of a sugarmoiety of the nucleoside.

A “nucleotide incorporating biocatalyst” refers to a catalyst thatcatalyzes the incorporation of nucleotides into a nucleic acid.Nucleotide incorporating biocatalysts are typically enzymes. An “enzyme”is a protein- and/or nucleic acid-based catalyst that acts to reduce theactivation energy of a chemical reaction involving other compounds or“substrates.” A “nucleotide incorporating enzyme” refers to an enzymethat catalyzes the incorporation of nucleotides into a nucleic acid.Exemplary nucleotide incorporating enzymes include, e.g., DNApolymerases, RNA polymerases, terminal transferases, reversetranscriptases, telomerases, polynucleotide phosphorylases, and thelike.

An “oligonucleotide” refers to a nucleic acid that includes at least twonucleic acid monomer units (e.g., nucleotides), typically more thanthree monomer units, and more typically greater than ten monomer units.The exact size of an oligonucleotide generally depends on variousfactors, including the ultimate function or use of the oligonucleotide.To further illustrate, oligonucleotides are typically less than 200residues long (e.g., between 15 and 100), however, as used herein, theterm is also intended to encompass longer polynucleotide chains.Oligonucleotides are often referred to by their length. For example a 24residue oligonucleotide is referred to as a “24-mer”. Typically, thenucleoside monomers are linked by phosphodiester bonds or analogsthereof, including phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like, including associatedcounterions, e.g., H⁺, NH₄′, Na⁺, and the like, if such counterions arepresent. Further, oligonucleotides are typically single-stranded.Oligonucleotides are optionally prepared by any suitable method,including, but not limited to, isolation of an existing or naturalsequence, DNA replication or amplification, reverse transcription,cloning and restriction digestion of appropriate sequences, or directchemical synthesis by a method such as the phosphotriester method ofNarang et al. (1979) Meth Enzymol. 68: 90-99; the phosphodiester methodof Brown et al. (1979) Meth Enzymol. 68: 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) TetrahedronLett. 22: 1859-1862; the triester method of Matteucci et al. (1981)J AmChem Soc. 103:3185-3191; automated synthesis methods; or the solidsupport method of U.S. Pat. No. 4,458,066, entitled “PROCESS FORPREPARING POLYNUCLEOTIDES,” issued Jul. 3, 1984 to Caruthers et al., orother methods known to those skilled in the art. All of these referencesare incorporated by reference.

As used herein a “sample” refers to anything capable of being analyzedby the methods provided herein. In some embodiments, the samplecomprises or is suspected to comprise one or more nucleic acids capableof analysis by the methods. Preferably, the samples comprise nucleicacids (e.g., DNA, RNA, cDNAs, etc.) from one or more bioagents. Samplescan include, for example, blood, saliva, urine, feces, anorectal swabs,vaginal swabs, cervical swabs, and the like. In some embodiments, thesamples are “mixture” samples, which comprise nucleic acids from morethan one subject or individual. In some embodiments, the methodsprovided herein comprise purifying the sample or purifying the nucleicacid(s) from the sample. In some embodiments, the sample is purifiednucleic acid.

A “sequence” of a biopolymer refers to the order and identity of monomerunits (e.g., nucleotides, etc.) in the biopolymer. The sequence (e.g.,base sequence) of a nucleic acid is typically read in the 5′ to 3′direction.

As is used herein, the term “single primer pair identification” meansthat one or more bioagents can be identified using a single primer pair.A base composition signature for an amplicon may singly identify one ormore bioagents.

A “solid support” refers to a solid material which can be derivatizedwith, or otherwise attached to, a chemical moiety, such as a primernucleic acid, a template nucleic acid, or the like. Exemplary solidsupports include a zero-mode waveguide array, a plate, a bead, amicrobead, a fiber, a whisker, a comb, a hybridization chip, a membrane,a single crystal, a ceramic layer, a self-assembling monolayer, and thelike.

As used herein, a “sub-species characteristic” is a geneticcharacteristic that provides the means to distinguish two members of thesame bioagent species. For example, one viral strain may bedistinguished from another viral strain of the same species bypossessing a genetic change (e.g., for example, a nucleotide deletion,addition or substitution) in one of the viral genes, such as theRNA-dependent RNA polymerase.

A “subsequence” or “fragment” refers to any portion of an entire nucleicacid sequence.

As used herein, in some embodiments the term “substantialcomplementarity” means that a primer member of a primer pair comprisesbetween about 70%-100%, or between about 80-100%, or between about90-100%, or between about 95-100%, or between about 99-100%complementarity with the conserved binding sequence of a nucleic acidfrom a given bioagent. These ranges of complementarity and identity areinclusive of all whole or partial numbers embraced within the recitedrange numbers. For example, and not limitation, 75.667%, 82%, 91.2435%and 97% complementarity or sequence identity are all numbers that fallwithin the above recited range of 70% to 100%, therefore forming a partof this description.

A “system” in the context of analytical instrumentation refers a groupof objects and/or devices that form a network for performing a desiredobjective.

A “template nucleic acid” refers to a nucleic acid to which a primernucleic acid can hybridize and be extended. Accordingly, templatenucleic acids include subsequences that are at least partiallycomplementary to the primer nucleic acids. Template nucleic acids can bederived from essentially any source. To illustrate, template nucleicacids are optionally derived or isolated from, e.g., culturedmicroorganisms, uncultured microorganisms, complex biological mixtures,tissues, sera, pooled sera or tissues, multispecies consortia, ancient,fossilized or other nonliving biological remains, environmentalisolates, soils, groundwaters, waste facilities, deep-sea environments,or the like. Further, template nucleic acids optionally include or arederived from, e.g., individual cDNA molecules, cloned sets of cDNAs,cDNA libraries, extracted RNAs, natural RNAs, in vitro transcribed RNAs,characterized or uncharacterized genomic DNAs, cloned genomic DNAs,genomic DNA libraries, enzymatically fragmented DNAs or RNAs, chemicallyfragmented DNAs or RNAs, physically fragmented DNAs or RNAs, or thelike. Template nucleic acids can also be chemically synthesized usingtechniques known in the art. In addition, template nucleic acidsoptionally correspond to at least a portion of a gene or arecomplementary thereto.

As used herein, “triangulation identification” means the use of morethan one primer pair to generate a corresponding amplicon foridentification of a bioagent. The more than one primer pair can be usedin individual wells or vessels or in a multiplex PCR assay.Alternatively, PCR reactions may be carried out in single wells orvessels comprising a different primer pair in each well or vessel.Following amplification the amplicons are pooled into a single well orcontainer which is then subjected to base composition analysis (e.g.,which does not involve molecular mass analysis). The combination ofpooled amplicons can be chosen such that the expected ranges of basecompositions of individual amplicons are not overlapping and thus willnot complicate identification of signals. Triangulation is a process ofelimination, wherein a first primer pair identifies that an unknownbioagent may be one of a group of bioagents. Subsequent primer pairs areused in triangulation identification to further refine the identity ofthe bioagent amongst the subset of possibilities generated with theearlier primer pair. Triangulation identification is complete when theidentity of the bioagent is determined. The triangulation identificationprocess may also be used to reduce false negative and false positivesignals, and enable reconstruction of the origin of hybrid or otherwiseengineered bioagents. For example, identification of the three parttoxin genes typical of B. anthracis (Bowen et al., J Appl Microbiol.,1999, 87, 270-278) in the absence of the expected compositions from theB. anthracis genome would suggest a genetic engineering event.

As used herein, the term “unknown bioagent” can mean, for example: (i) abioagent whose existence is not known (for example, the SARS coronaviruswas unknown prior to April 2003) and/or (ii) a bioagent whose existenceis known (such as the well known bacterial species Staphylococcus aureusfor example) but which is not known to be in a sample to be analyzed.For example, if the method for identification of coronaviruses disclosedin commonly owned U.S. patent Ser. No. 10/829,826 (incorporated hereinby reference in its entirety) was to be employed prior to April 2003 toidentify the SARS coronavirus in a clinical sample, both meanings of“unknown” bioagent are applicable since the SARS coronavirus was unknownto science prior to April, 2003 and since it was not known what bioagent(in this case a coronavirus) was present in the sample. On the otherhand, if the method of U.S. patent Ser. No. 10/829,826 was to beemployed subsequent to April 2003 to identify the SARS coronavirus in aclinical sample, the second meaning (ii) of “unknown” bioagent wouldapply because the SARS coronavirus became known to science subsequent toApril 2003 because it was not known what bioagent was present in thesample.

As used herein, the term “variable region” is used to describe a regionthat falls between any one primer pair described herein. The regionpossesses distinct base compositions between at least two bioagents,such that at least one bioagent can be identified at, for example, thefamily, genus, species or sub-species level. The degree of variabilitybetween the at least two bioagents need only be sufficient to allow foridentification using mass spectrometry analysis, as described herein.

As used herein, a “wobble base” is a variation in a codon found at thethird nucleotide position of a DNA triplet. Variations in conservedregions of sequence are often found at the third nucleotide position dueto redundancy in the amino acid code.

In certain embodiments, provided herein are methods, compositions, kits,and related systems for the detection and identification of bioagents(e.g., species of HPV) using bioagent identifying amplicons. In someembodiments, primers are selected to hybridize to conserved sequenceregions of nucleic acids derived from a bioagent and which flankvariable sequence regions to yield a bioagent identifying amplicon whichcan be amplified and which is amenable to base composition analysis. Insome embodiments, the corresponding base composition of one or moredifferent amplicons is queried against a database of base compositionsindexed to bioagents and to the primer pair used to generate theamplicon. A match of the measured base composition to a database entrybase composition associates the sample bioagent to an indexed bioagentin the database. Thus, the identity of the unknown bioagent isdetermined. No prior knowledge of the unknown bioagent is necessary tomake an identification. In some instances, the measured base compositionassociates with more than one database entry base composition. Thus, asecond/subsequent primer pair is generally used to generate an amplicon,and its measured base composition is similarly compared to the databaseto determine its identity in triangulation identification. Furthermore,the methods and other aspects of the invention can be applied to rapidparallel multiplex analyses, the results of which can be employed in atriangulation identification strategy. Thus, in some embodiments, thepresent invention provides rapid throughput and does not require nucleicacid sequencing or knowledge of the linear sequences of nucleobases ofthe amplified target sequence for bioagent detection and identification.

Methods of employing base compositions, databases containing basecomposition entries, and triangulation using primers, are described inthe following patents, patent applications and scientific publications,all of which are herein incorporated by reference as if fully set forthherein: U.S. Pat. Nos. 7,108,974; 7,217,510; 7,226,739; 7,255,992;7,312,036; 7,339,051; US patent publication numbers 2003/0027135;2003/0167133; 2003/0167134; 2003/0175695; 2003/0175696; 2003/0175697;2003/0187588; 2003/0187593; 2003/0190605; 2003/0225529; 2003/0228571;2004/0110169; 2004/0117129; 2004/0121309; 2004/0121310; 2004/0121311;2004/0121312; 2004/0121313; 2004/0121314; 2004/0121315; 2004/0121329;2004/0121335; 2004/0121340; 2004/0122598; 2004/0122857; 2004/0161770;2004/0185438; 2004/0202997; 2004/0209260; 2004/0219517; 2004/0253583;2004/0253619; 2005/0027459; 2005/0123952; 2005/0130196 2005/0142581;2005/0164215; 2005/0266397; 2005/0270191; 2006/0014154; 2006/0121520;2006/0205040; 2006/0240412; 2006/0259249; 2006/0275749; 2006/0275788;2007/0087336; 2007/0087337; 2007/0087338 2007/0087339; 2007/0087340;2007/0087341; 2007/0184434; 2007/0218467; 2007/0218467; 2007/0218489;2007/0224614; 2007/0238116; 2007/0243544; 2007/0248969; WO2002/070664;WO2003/001976; WO2003/100035; WO2004/009849; WO2004/052175;WO2004/053076; WO2004/053141; WO2004/053164; WO2004/060278;WO2004/093644; WO 2004/101809; WO2004/111187; WO2005/023083;WO2005/023986; WO2005/024046; WO2005/033271; WO2005/036369;WO2005/086634; WO2005/089128; WO2005/091971; WO2005/092059;WO2005/094421; WO2005/098047; WO2005/116263; WO2005/117270;WO2006/019784; WO2006/034294; WO2006/071241; WO2006/094238;WO2006/116127; WO2006/135400; WO2007/014045; WO2007/047778;WO2007/086904; WO2007/100397; and WO2007/118222, all of which are hereinincorporated by reference.

Exemplary base-count related methods and other aspects of use in themethods, systems, and other aspects of the invention are also describedin, for example, Ecker et al., Ibis T5000: a universal biosensorapproach for microbiology. Nat Rev Microbiol. 2008 Jun. 3.; Ecker etal., The Microbial Rosetta Stone Database: A compilation of global andemerging infectious microorganisms and bioterrorist threat agents.;Ecker et al., The Ibis T5000 Universal Biosensor: An Automated Platformfor Pathogen Identification and Strain Typing.; Ecker et al., TheMicrobial Rosetta Stone Database: A common structure for microbialbiosecurity threat agents.; Ecker et al., Identification ofAcinetobacter species and genotyping of Acinetobacter baumannii bymultilocus PCR and mass spectrometry. J Clin Microbiol. 2006 August;44(8):2921-32.; Ecker et al., Rapid identification and strain-typing ofrespiratory pathogens for epidemic surveillance. Proc Natl Acad Sci USA.2005 May 31; 102(22):8012-7. Epub 2005 May 23.; Wortmann et al.,Genotypic evolution of Acinetobacter baumannii Strains in an outbreakassociated with war trauma, Infect Control Hosp Epidemiol. 2008 June;29(6):553-555.; Hannis et al., High-resolution genotyping ofCampylobacter species by use of PCR and high-throughput massspectrometry. J Clin Microbiol. 2008 April; 46(4): 1220-5.; Blyn et al.,Rapid detection and molecular serotyping of adenovirus by use of PCRfollowed by electrospray ionization mass spectrometry. J Clin Microbiol.2008 February; 46(2):644-51.; Eshoo et al., Direct broad-range detectionof alphaviruses in mosquito extracts, Virology. 2007 Nov. 25;368(2):286-95.; Sampath et al., Global surveillance of emergingInfluenza virus genotypes by mass spectrometry. PLoS ONE. 2007 May 30;2(5):e489.; Sampath et al., Rapid identification of emerging infectiousagents using PCR and electrospray ionization mass spectrometry. Ann NYAcad Sci. 2007 April; 1102: 109-20.; Hujer et al., Analysis ofantibiotic resistance genes in multidrug-resistant Acinetobacter sp.isolates from military and civilian patients treated at the Walter ReedArmy Medical Center. Antimicrob Agents Chemother. 2006 December;50(12):4114-23.; Hall et al., Base composition analysis of humanmitochondrial DNA using electrospray ionization mass spectrometry: anovel tool for the identification and differentiation of humans. AnalBiochem. 2005 Sep. 1; 344(1):53-69.; Sampath et al., Rapididentification of emerging pathogens: coronavirus. Emerg Infect Dis.2005 March; 11(3):373-9.; Jiang Y, Hofstadler S A. A highly efficientand automated method of purifying and desalting PCR products foranalysis by electrospray ionization mass spectrometry; Jiang et al.,Mitochondrial DNA mutation detection by electrospray mass spectrometry;Russell et al., Transmission dynamics and prospective environmentalsampling of adenovirus in a military recruit setting; Hofstadler et al.,Detection of microbial agents using broad-range PCR with detection bymass spectrometry: The TIGER concept. Chapter in; Hofstadler et al.,Selective ion filtering by digital thresholding: A method to unwindcomplex ESI-mass spectra and eliminate signals from low molecular weightchemical noise.; Hofstadler et al., TIGER: The Universal Biosensor.; VanErt et al., Mass spectrometry provides accurate characterization of twogenetic marker types in Bacillus anthracis.; Sampath et al., Forum onMicrobial Threats: Learning from SARS: Preparing for the Next DiseaseOutbreak—Workshop Summary (ed. Knobler S E, Mahmoud A, Lemon S.) TheNational Academies Press, Washington, D.C. 2004. 181-185.

In some embodiments, amplicons corresponding to bioagent identifyingamplicons are obtained using the polymerase chain reaction (PCR). Otheramplification methods may be used such as ligase chain reaction (LCR),low-stringency single primer PCR, and multiple strand displacementamplification (MDA). (Michael, S F., Biotechniques (1994), 16:411-412and Dean et al., Proc. Natl. Acad. Sci. U.S.A. (2002), 99, 5261-5266).

One embodiment of a process flow diagram used for primer selection andvalidation process is depicted in FIGS. 4 and 5. For each group oforganisms, candidate target sequences are identified (200) from whichnucleotide sequence alignments are created (210) and analyzed (220).Primers are then configured by selecting priming regions (230) tofacilitate the selection of candidate primer pairs (240). The primerpair sequence is typically a “best fit” amongst the aligned sequences,such that the primer pair sequence may or may not be fully complementaryto the hybridization region on any one of the bioagents in thealignment. Thus, best fit primer pair sequences are those withsufficient complementarity with two or more bioagents to hybridize withthe two or more bioagents and generate an amplicon. The primer pairs arethen subjected to in silico analysis by electronic PCR (ePCR) (300)wherein bioagent identifying amplicons are obtained from sequencedatabases such as GenBank or other sequence collections (310) and testedfor specificity in silico (320). Bioagent identifying amplicons obtainedfrom ePCR of GenBank sequences (310) may also be analyzed by aprobability model which predicts the capability of a given amplicon toidentify unknown bioagents. Preferably, the base compositions ofamplicons with favorable probability scores are then stored in a basecomposition database (325). Alternatively, base compositions of thebioagent identifying amplicons obtained from the primers and GenBanksequences are directly entered into the base composition database (330).Candidate primer pairs (240) are validated by in vitro amplification bya method such as PCR analysis (400) of nucleic acid from a collection oforganisms (410). Amplicons thus obtained are analyzed to confirm thesensitivity, specificity and reproducibility of the primers used toobtain the amplicons (420).

Synthesis of primers is well known and routine in the art. The primersmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed.

The primers, in some embodiments, are employed as compositions for usein methods for identification of bioagents as follows: a primer paircomposition is contacted with nucleic acid of an unknown isolatesuspected of comprising a target bioagent. The nucleic acid is thenamplified by a nucleic acid amplification technique, such as PCR forexample, to obtain an amplicon that represents a bioagent identifyingamplicon. The base composition of the double-stranded amplicon, orsingle strand corresponding to only the forward or reverse primer, isdetermined by techniques such as sequencing, HPLC, and paperchromatography (see, e.g., Voelkerding et al., Clinical Chem.,“Next-generation sequencing: from basic research to diagnostics,” 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296, andManderville and Kropinski, “Approaches to the Compositional Analysis ofDNA,” Methods Mol Biol. 2009; 502:11-7, all of which are hereinincorporated by reference). A measured base composition calculatedtherefrom is then compared with a database of base compositions indexedto primer pairs and to known bioagents. A match between the basecomposition of the amplicon and the database base composition for thatindexed primer pair correlates the measured base composition with anindexed bioagent, thus identifying the unknown bioagent (e.g. thespecies of virus). In some embodiments, the method is repeated using adifferent primer pair to resolve possible ambiguities in theidentification process or to improve the confidence level for theidentification assignment (triangulation identification). In someembodiments, for example, where the unknown is a novel, previouslyuncharacterized organism, the base composition from an amplicongenerated from the unknown is matched with one or more best match basecompositions from a database to predict a family, genus, species,sub-type, etc. of the unknown. Such information may assist furthercharacterization of the unknown or provide a physician treating apatient infected by the unknown with a therapeutic agent best calculatedto treat the patient.

In certain embodiments, the bioagent is detected with the systems andmethods of the present invention in combination with other bioagents,including viruses, bacteria, fungi, or other bioagents. In particularembodiments, a panel is employed that includes a first bioagent andother related or un-related bioagents. Such panels may be specific for aparticular type of bioagent, or specific for a specific type of test(e.g., for testing the safety of blood, one may include commonly presentviral pathogens such as HCV, HIV, and bacteria that can be contractedvia a blood transfusion).

In some embodiments, a bioagent identifying amplicon may be producedusing only a single primer (either the forward or reverse primer of anygiven primer pair), provided an appropriate amplification method ischosen, such as, for example, low stringency single primer PCR(LSSP-PCR).

In some embodiments, the oligonucleotide primers are broad range surveyprimers which hybridize to conserved regions of nucleic acid. The broadrange primer may identify the unknown bioagent depending on whichbioagent is in the sample. In other cases, the base composition of anamplicon does not provide sufficient resolution to identify the unknownbioagent as any one bioagent at or below the species level. These casesgenerally benefit from further analysis of one or more ampliconsgenerated from at least one additional broad range survey primer pair,or from at least one additional division-wide primer pair, or from atleast one additional drill-down primer pair. Identification ofsub-species characteristics may be required, for example, to determine aclinical treatment of patient, or in rapidly responding to an outbreakof a new species, sub-type, etc. of pathogen to prevent an epidemic orpandemic.

One with ordinary skill in the art of design of amplification primerswill recognize that a given primer need not hybridize with 100%complementarity in order to effectively prime the synthesis of acomplementary nucleic acid strand in an amplification reaction. Primerpair sequences may be a “best fit” amongst the aligned bioagentsequences, thus they need not be fully complementary to thehybridization region of any one of the bioagents in the alignment.Moreover, a primer may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridizationevent (e.g., for example, a loop structure or a hairpin structure).Thus, in some embodiments, an extent of variation of 70% to 100%, or anyrange falling within, of the sequence identity is possible relative tothe specific primer sequences disclosed herein. To illustrate,determination of sequence identity is described in the followingexample: a primer 20 nucleobases in length which is identical to another20 nucleobase primer having two non-identical residues has 18 of 20identical residues (18/20=0.9 or 90% sequence identity). In anotherexample, a primer 15 nucleobases in length having all residues identicalto a 15 nucleobase segment of primer 20 nucleobases in length would have15/20=0.75 or 75% sequence identity with the 20 nucleobase primer.Percent identity need not be a whole number, for example when a 28consecutive nucleobase primer is completely identical to a 31consecutive nucleobase primer (28/31=0.9032 or 90.3% identical).

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome embodiments, complementarity of primers with respect to theconserved priming regions of viral nucleic acid, is between about 70%and about 80%. In other embodiments, homology, sequence identity orcomplementarity, is between about 80% and about 90%. In yet otherembodiments, homology, sequence identity or complementarity, is at least90%, at least 92%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or is 100%.

In some embodiments, the primers described herein comprise at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or100% (or any range falling within) sequence identity with the primersequences specifically disclosed herein.

In some embodiments, the oligonucleotide primers are 13 to 35nucleobases in length (13 to 35 linked nucleotide residues). Theseembodiments comprise oligonucleotide primers 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35nucleobases in length, or any range therewithin.

In some embodiments, any given primer comprises a modificationcomprising the addition of a non-templated T residue to the 5′ end ofthe primer (i.e., the added T residue does not necessarily hybridize tothe nucleic acid being amplified). The addition of a non-templated Tresidue has an effect of minimizing the addition of non-templated Aresidues as a result of the non-specific enzyme activity of, e.g., TaqDNA polymerase (Magnuson et al., Biotechniques, 1996, 21, 700-709), anoccurrence which may lead to ambiguous results arising from molecularmass analysis.

Primers may contain one or more universal bases. Because any variation(due to codon wobble in the third position) in the conserved regionsamong species is likely to occur in the third position of a DNA (or RNA)triplet, oligonucleotide primers can be designed such that thenucleotide corresponding to this position is a base which can bind tomore than one nucleotide, referred to herein as a “universalnucleobase.” For example, under this “wobble” base pairing, inosine (I)binds to U, C or A; guanine (G) binds to U or C, and uridine (U) bindsto U or C. Other examples of universal nucleobases include nitroindolessuch as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides andNucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK,an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot etal., Nucleosides and Nucleotides., 1995, 14, 1053-1056) or the purineanalog 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide (Sala etal., Nucl. Acids Res., 1996, 24, 3302-3306).

In some embodiments, to compensate for weaker binding by the wobblebase, oligonucleotide primers are configured such that the first andsecond positions of each triplet are occupied by nucleotide analogswhich bind with greater affinity than the unmodified nucleotide.Examples of these analogs include, but are not limited to,2,6-diaminopurine which binds to thymine, 5-propynyluracil which bindsto adenine and 5-propynylcytosine and phenoxazines, including G-clamp,which binds to G. Propynylated pyrimidines are described in U.S. Pat.Nos. 5,645,985, 5,830,653 and 5,484,908, each of which is commonly ownedand incorporated herein by reference in its entirety. Propynylatedprimers are described in U.S Pre-Grant Publication No. 2003-0170682;also commonly owned and incorporated herein by reference in itsentirety. Phenoxazines are described in U.S. Pat. Nos. 5,502,177,5,763,588, and 6,005,096, each of which is incorporated herein byreference in its entirety. G-clamps are described in U.S. Pat. Nos.6,007,992 and 6,028,183, each of which is incorporated herein byreference in its entirety.

In some embodiments, non-template primer tags are used to increase themelting temperature (T_(m)) of a primer-template duplex in order toimprove amplification efficiency. A non-template tag is at least threeconsecutive A or T nucleotide residues on a primer which are notcomplementary to the template. In any given non-template tag, A can bereplaced by C or G and T can also be replaced by C or G. AlthoughWatson-Crick hybridization is not expected to occur for a non-templatetag relative to the template, the extra hydrogen bond in a G-C pairrelative to an A-T pair confers increased stability of theprimer-template duplex and improves amplification efficiency forsubsequent cycles of amplification when the primers hybridize to strandssynthesized in previous cycles.

In other embodiments, propynylated tags may be used in a manner similarto that of the non-template tag, wherein two or more 5-propynylcytidineor 5-propynyluridine residues replace template matching residues on aprimer. In other embodiments, a primer contains a modifiedinternucleoside linkage such as a phosphorothioate linkage, for example.

In some embodiments, the primers contain mass-modifying tags. Reducingthe total number of possible base compositions of a nucleic acid ofspecific molecular weight provides a means of avoiding a possible sourceof ambiguity in the determination of base composition of amplicons.

In some embodiments, assignment of previously unobserved basecompositions (also known as “true unknown base compositions”) to a givenphylogeny can be accomplished via the use of pattern classifier modelalgorithms. Base compositions, like sequences, may vary slightly fromstrain to strain within species, for example. In some embodiments, thepattern classifier model is the mutational probability model. In otherembodiments, the pattern classifier is the polytope model. A polytopemodel is the mutational probability model that incorporates both therestrictions among strains and position dependence of a given nucleobasewithin a triplet. In certain embodiments, a polytope pattern classifieris used to classify a test or unknown organism according to its ampliconbase composition.

In some embodiments, it is possible to manage this diversity by building“base composition probability clouds” around the composition constraintsfor each species. A “pseudo four-dimensional plot” may be used tovisualize the concept of base composition probability clouds. Optimalprimer design typically involves an optimal choice of bioagentidentifying amplicons and maximizes the separation between the basecomposition signatures of individual bioagents. Areas where cloudsoverlap generally indicate regions that may result in amisclassification, a problem which is overcome by a triangulationidentification process using bioagent identifying amplicons not affectedby overlap of base composition probability clouds.

In some embodiments, base composition probability clouds provide themeans for screening potential primer pairs in order to avoid potentialmisclassifications of base compositions. In other embodiments, basecomposition probability clouds provide the means for predicting theidentity of an unknown bioagent whose assigned base composition has notbeen previously observed and/or indexed in a bioagent identifyingamplicon base composition database due to evolutionary transitions inits nucleic acid sequence.

Provided herein is bioagent classifying information at a levelsufficient to identify a given bioagent. Furthermore, the process ofdetermining a previously unknown base composition for a given bioagent(for example, in a case where sequence information is unavailable) hasutility by providing additional bioagent indexing information with whichto populate base composition databases. The process of future bioagentidentification is thus improved as additional base composition signatureindexes become available in base composition databases.

In certain embodiments, a sample comprising an unknown bioagent iscontacted with a primer pair which amplifies the nucleic acid from thebioagent, and a known quantity of a polynucleotide that comprises acalibration sequence. The amplification reaction then produces twoamplicons: a bioagent identifying amplicon and a calibration amplicon.The bioagent identifying amplicon and the calibration amplicon aredistinguishable by base composition while being amplified at essentiallythe same rate. Effecting differential base compositions can beaccomplished by choosing as a calibration sequence, a representativebioagent identifying amplicon (from a specific species of bioagent) andperforming, for example, a 2-8 nucleobase deletion or insertion withinthe variable region between the two priming sites, a calibrationsequence with a different base composition due to base substitutions.The amplified sample containing the bioagent identifying amplicon andthe calibration amplicon is then subjected to base composition analysis(e.g., sequencing, HPLC, paper chromatography, etc.) without determiningthe molecular mass of the sequences. The resulting base compositionanalysis of the nucleic acid of the bioagent and of the calibrationsequence provides base composition data and abundance data for thenucleic acid of the bioagent and of the calibration sequence. The basecomposition data obtained for the nucleic acid of the bioagent enablesidentification of the unknown bioagent by base composition analysis. Theabundance data enables calculation of the quantity of the bioagent,based on the knowledge of the quantity of calibration polynucleotidecontacted with the sample.

In some embodiments, construction of a standard curve in which theamount of calibration or calibrant polynucleotide spiked into the sampleis varied provides additional resolution and improved confidence for thedetermination of the quantity of bioagent in the sample. Alternatively,the calibration polynucleotide can be amplified in its own reactionvessel or vessels under the same conditions as the bioagent. A standardcurve may be prepared there from, and the relative abundance of thebioagent determined by methods such as linear regression. In someembodiments, multiplex amplification is performed where multiplebioagent identifying amplicons are amplified with multiple primer pairswhich also amplify the corresponding standard calibration sequences. Inthis or other embodiments, the standard calibration sequences areoptionally included within a single construct (preferably a vector)which functions as the calibration polynucleotide.

In some embodiments, the calibrant polynucleotide is used as an internalpositive control to confirm that amplification conditions and subsequentanalysis steps are successful in producing a measurable amplicon. Evenin the absence of copies of the genome of a bioagent, the calibrationpolynucleotide gives rise to a calibration amplicon. Failure to producea measurable calibration amplicon indicates a failure of amplificationor subsequent analysis step such as amplicon purification or basecomposition determination. Reaching a conclusion that such failures haveoccurred is, in itself, a useful event. In some embodiments, thecalibration sequence is comprised of DNA. In some embodiments, thecalibration sequence is comprised of RNA.

In some embodiments, a calibration sequence is inserted into a vectorwhich then functions as the calibration polynucleotide. In someembodiments, more than one calibration sequence is inserted into thevector that functions as the calibration polynucleotide. Such acalibration polynucleotide is herein termed a “combination calibrationpolynucleotide.” It should be recognized that the calibration methodshould not be limited to the embodiments described herein. Thecalibration method can be applied for determination of the quantity ofany bioagent identifying amplicon when an appropriate standard calibrantpolynucleotide sequence is designed and used.

In certain embodiments, primer pairs are configured to produce bioagentidentifying amplicons within more conserved regions of a bioagent, whileothers produce bioagent identifying amplicons within regions that aremay evolve more quickly. Primer pairs that characterize amplicons in aconserved region with low probability that the region will evolve pastthe point of primer recognition are useful, e.g., as a broad rangesurvey-type primer. Primer pairs that characterize an ampliconcorresponding to an evolving genomic region are useful, e.g., fordistinguishing emerging bioagent strain variants.

The primer pairs described herein provide reagents, e.g., foridentifying diseases caused by emerging types of bioagents. Basecomposition analysis eliminates the need for prior knowledge of bioagentsequence to generate hybridization probes. Thus, in another embodiment,there is provided a method for determining the etiology of a particularstain when the process of identification of is carried out in a clinicalsetting, and even when a new strain is involved. This is possiblebecause the methods may not be confounded by naturally occurringevolutionary variations.

Another embodiment provides a means of tracking the spread of anyspecies or strain of particular bioagents when a plurality of samplesobtained from different geographical locations are analyzed by methodsdescribed above in an epidemiological setting. For example, a pluralityof samples from a plurality of different locations may be analyzed withprimers which produce bioagent identifying amplicons, a subset of whichidentifies a specific strain. The corresponding locations of the membersof the strain-containing subset indicate the spread of the specificstrain to the corresponding locations.

Also provided are kits for carrying out the methods described herein. Insome embodiments, the kit may comprise a sufficient quantity of one ormore primer pairs to perform an amplification reaction on a targetpolynucleotide from a bioagent to form a bioagent identifying amplicon.In some embodiments, the kit may comprise from one to twenty primerpairs, from one to ten primer pairs, from one to eight pairs, from oneto five primer pairs, from one to three primer pairs, or from one to twoprimer pairs.

In some embodiments, the kit may also comprise a sufficient quantity ofreverse transcriptase, a DNA polymerase, suitable nucleosidetriphosphates (including any of those described above), a DNA ligase,and/or reaction buffer, or any combination thereof, for theamplification processes described above. The kit may also comprisereagents necessary for performing sequencing methods, or HPLC or paperchromatography (see, e.g., Voelkerding et al., Clinical Chem.,“Next-generation sequencing: from basic research to diagnostics,” 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296, andManderville and Kropinski, “Approaches to the Compositional Analysis ofDNA,” Methods Mol Biol. 2009; 502:11-7, all of which are hereinincorporated by reference).

A kit may further include instructions pertinent for the particularembodiment of the kit, such instructions describing the primer pairs andamplification conditions for operation of the method. In someembodiments, the kit further comprises instructions for analysis,interpretation and dissemination of data acquired by the kit. In otherembodiments, instructions for the operation, analysis, interpretationand dissemination of the data of the kit are provided on computerreadable media. A kit may also comprise amplification reactioncontainers such as microcentrifuge tubes, microtiter plates, and thelike. A kit may also comprise reagents or other materials for isolatingbioagent nucleic acid or bioagent identifying amplicons fromamplification reactions, including, for example, detergents, solvents,or ion exchange resins which may be linked to magnetic beads. A kit mayalso comprise a table of measured or calculated base compositions ofbioagents using the primer pairs of the kit.

The invention also provides systems that can be used to perform variousassays relating to bioagent detection or identification. In certainembodiments, systems include sequencing devices (or HPLC equipment orpaper chromatography equipment) configured to detect base compositionsof amplicons produced using purified oligonucleotide primer pairsdescribed herein. Other devices/equipment that are optionally adaptedfor use in the systems of the invention are described further below. Insome embodiments, systems also include controllers operably connected tosequencing devices and/or other system components. In some of theseembodiments, controllers are configured to correlate the basecompositions of the amplicons with bioagents to effect detection oridentification. As described herein, the base compositions generallycorrespond to the bioagent species identities. In certain embodiments,controllers include, or are operably connected to, databases of knownbase compositions of amplicons of known species of bioagents producedwith the primer pairs described herein. Controllers are describedfurther below.

In certain embodiments, the oligonucleotides are arrayed on solidsupports, whereas in others, they are provided in one or morecontainers, e.g., for assays performed in solution. In certainembodiments, the systems also include at least one detector or detectioncomponent (e.g., a spectrometer) that is configured to detect detectablesignals produced in the container or on the support. In addition, thesystems also optionally include at least one thermal modulator (e.g., athermal cycling device) operably connected to the containers or solidsupports to modulate temperature in the containers or on the solidsupports, and/or at least one fluid transfer component (e.g., anautomated pipettor) that transfers fluid to and/or from the containersor solid supports, e.g., for performing one or more assays (e.g.,nucleic acid amplification, real-time amplicon detection, etc.) in thecontainers or on the solid supports.

Examples of suitable thermocycling devices that are optionally utilizedare available from many different commercial suppliers, includingMastercycler® devices (Eppendorf North America, Westbury, N.Y., U.S.A.),the COBAS® AMPLICOR Analyzer (Roche Molecular Systems, Inc., Pleasanton,Calif., U.S.A.), MyCycler and iCycler Thermal Cyclers (Bio-RadLaboratories, Inc., Hercules, Calif., U.S.A.), and the SmartCyclerSystem (Cepheid, Sunnyvale, Calif. U.S.A.), among many others. In otherexemplary embodiments, sample preparation components, nucleic acidamplification components, and related fluid handling or materialtransfer components are integrated with the systems described herein,e.g., to fully automate a given nucleic acid amplification and analysisprocess. Instruments that can be adapted for this purpose include, forexample, the m2000™ automated instrument system (Abbott Laboratories,Abbott Park, Ill., U.S.A.), the GeneXpert System (Cepheid, Sunnyvale,Calif. U.S.A.), and the COBAS® AmpliPrep® System (Roche MolecularSystems, Inc., Pleasanton, Calif., U.S.A.), and the like.

Detectors are typically structured to detect detectable signalsproduced, e.g., in or proximal to another component of the given assaysystem (e.g., in a container and/or on a solid support). Suitable signaldetectors that are optionally utilized, or adapted for use, hereindetect, e.g., fluorescence, phosphorescence, radioactivity, absorbance,refractive index, luminescence, or mass. Detectors optionally monitorone or a plurality of signals from upstream and/or downstream of theperformance of, e.g., a given assay step. For example, detectorsoptionally monitor a plurality of optical signals, which correspond inposition to “real-time” results. Example detectors or sensors includephotomultiplier tubes, CCD arrays, optical sensors, temperature sensors,pressure sensors, pH sensors, conductivity sensors, or scanningdetectors. Detectors are also described in, e.g., Skoog et al.,Principles of Instrumental Analysis, 6^(th) Ed., Brooks Cole (2006),Currell, Analytical

Instrumentation: Performance Characteristics and Quality, John Wiley &Sons, Inc. (2000), Sharma et al., Introduction to FluorescenceSpectroscopy, John Wiley & Sons, Inc. (1999), Valeur, MolecularFluorescence: Principles and Applications, John Wiley & Sons, Inc.(2002), and Gore, Spectrophotometry and Spectrofluorimetry: A PracticalApproach, 2^(nd) Ed., Oxford University Press (2000), which are eachincorporated by reference.

As mentioned above, the systems of the invention also typically includecontrollers that are operably connected to one or more components (e.g.,detectors, databases, thermal modulators, fluid transfer components,robotic material handling devices, and the like) of the given system tocontrol operation of the components. More specifically, controllers aregenerally included either as separate or integral system components thatare utilized, e.g., to receive data from detectors to effect and/orregulate temperature in the containers, or to effect and/or regulatefluid flow to or from selected containers. Controllers and/or othersystem components are optionally coupled to an appropriately programmedprocessor, computer, digital device, information appliance, or otherlogic device (e.g., including an analog to digital or digital to analogconverter as needed), which functions to instruct the operation of theseinstruments in accordance with preprogrammed or user input instructions,receive data and information from these instruments, and interpret,manipulate and report this information to the user. Suitable controllersare generally known in the art and are available from various commercialsources.

Any controller or computer optionally includes a monitor, which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display or liquid crystal display), or others.Computer circuitry is often placed in a box, which includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive such as awriteable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser. These components are illustrated further below.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set of parameterfields, e.g., in a graphic user interface (GUI), or in the form ofpreprogrammed instructions, e.g., preprogrammed for a variety ofdifferent specific operations. The software then converts theseinstructions to appropriate language for instructing the operation ofone or more controllers to carry out the desired operation. The computerthen receives the data from, e.g., sensors/detectors included within thesystem, and interprets the data, either provides it in a user understoodformat, or uses that data to initiate further controller instructions,in accordance with the programming.

FIG. 6 is a schematic showing a representative system that includes alogic device in which various aspects of the present invention may beembodied. As will be understood by practitioners in the art from theteachings provided herein, aspects of the invention are optionallyimplemented in hardware and/or software. In some embodiments, differentaspects of the invention are implemented in either client-side logic orserver-side logic. As will be understood in the art, the invention orcomponents thereof may be embodied in a media program component (e.g., afixed media component) containing logic instructions and/or data that,when loaded into an appropriately configured computing device, causethat device to perform as desired. As will also be understood in theart, a fixed media containing logic instructions may be delivered to aviewer on a fixed media for physically loading into a viewer's computeror a fixed media containing logic instructions may reside on a remoteserver that a viewer accesses through a communication medium in order todownload a program component.

More specifically, FIG. 6 schematically illustrates computer 1000 towhich sequencing device or system 1002 (e.g., SMRT detection array fromPacific Biosciences), fluid transfer component 1004 (e.g., a sampleinjection needle or the like), and database 1008 are operably connected.Optionally, one or more of these components are operably connected tocomputer 1000 via a server (not shown in FIG. 6). During operation,fluid transfer component 1004 typically transfers reaction mixtures orcomponents thereof (e.g., aliquots comprising amplicons) from multi-wellcontainer 1006 to sequencing device. Sequencing device 1002 then detectsthe nucleic acid sequence of the amplicons. Computer 1000 then typicallyreceives this sequence data, calculates base compositions from thisdata, and compares it with entries in database 1008 to identify speciesor strains of bioagents in a given sample. It will be apparent to one ofskill in the art that one or more components of the system schematicallydepicted in FIG. 6 are optionally fabricated integral with one another(e.g., in the same housing).

While the present invention has been described with specificity inaccordance with certain of its embodiments, the following examples serveonly to illustrate the invention and are not intended to limit the same.In order that the invention disclosed herein may be more efficientlyunderstood, examples are provided below. It should be understood thatthese examples are for illustrative purposes only and are not to beconstrued as limiting the invention in any manner.

Example 1 Non-Mass Determined Base Composition Analysis for TargetSequence Detection

This example illustrates that pathogens can be identified anddistinguished from one another using complete or partial basecomposition or count information without the need for mass determinationof bases. For example, Table I shows base composition (single strand)results for 16S_(—)1100-1188 (16S rRNA) primer amplification reactionsfor different species of bacteria. Such base composition data can begenerated by any type of sequencing methodology or system (see, e.g.,Voelkerding et al., Clinical Chem., “Next-generation sequencing: frombasic research to diagnostics,” 55: 641-658, 2009; and MacLean et al.,Nature Rev. Microbiol., 7: 287-296). Species which are repeated in thetable (e.g., Clostridium botulinum) are different strains which havedifferent base compositions in the 16S_(—)1100-1188 region. As shown inTable I, for example, a complete base composition using this primeramplification reaction will uniquely identify and distinguishMycobacterium avium (i.e., A₁₆G₃₂C₁₈T₁₆) from the other organisms listedin Table I, as will various partial base compositions using the sameprimer amplification reaction (e.g., A₁₆G₃₂, A₁₆C₁₈T₁₆, G₃₂C₁₈T₁₆,C₁₈T₁₆, etc.).

TABLE I Organism name Base comp. Organism name Base comp. MycobacteriumA₁₆G₃₂C₁₈T₁₆ Vibrio cholerae A₂₃G₃₀C₂₁T₁₆ avium Streptomyces sp.A₁₇G₃₈C₂₇T₁₄ Aeromonas hydrophila A₂₃G₃₁C₂₁T₁₅ Ureaplasma A₁₈G₃₀C₁₇T₁₇Aeromonas A₂₃G₃₁C₂₁T₁₅ urealyticum salmonicida Streptomyces sp.A₁₉G₃₆C₂₄T₁₈ Mycoplasma genitalium A₂₄G₁₉C₁₂T₁₈ MycobacteriumA₂₀G₃₂C₂₂T₁₆ Clostridium botulinum A₂₄G₂₅C₁₈T₂₀ leprae M. tuberculosisA₂₀G₃₃C₂₁T₁₆ Bordetella A₂₄G₂₆C₁₉T₁₄ bronchiseptica NocardiaA₂₀G₃₃C₂₁T₁₆ Francisella tularensis A₂₄G₂₆C₁₉T₁₉ asteroidesFusobacterium A₂₁G₂₆C₂₃T₁₆ Bacillus anthracis A₂₄G₂₆C₂₀T₁₈ necroforumListeria A₂₁G₂₇C₁₉T₁₉ Campylobacter jejuni A₂₄G₂₆C₂₀T₁₈ monocytogenesClostridium A₂₁G₂₇C₁₉T₂₁ Staphylococcus aureus A₂₄G₂₆C₂₀T₁₈ botulinumNeisseria A₂₁G₂₈C₂₁T₁₈ Helicobacter pylori A₂₄G₂₆C₂₀T₁₉ gonorrhoeaeBartonella A₂₁G₃₀C₂₂T₁₆ Helicobacter pylori A₂₄G₂₆C₂₁T₁₈ quintanaEnterococcus A₂₂G₂₇C₂₀T₁₉ Moraxella catarrhalis A₂₄G₂₆C₂₃T₁₆ faecalisBacillus A₂₂G₂₈C₂₀T₁₈ Haemophilus A₂₄G₂₈C₂₀T₁₇ megaterium influenzae RdBacillus subtilis A₂₂G₂₈C₂₁T₁₇ Chlamydia trachomatis A₂₄G₂₈C₂₁T₁₆Pseudomonas A₂₂G₂₉C₂₃T₁₅ Chlamydophila A₂₄G₂₈C₂₁T₁₆ aeruginosapneumoniae Legionella A₂₂G₃₂C₂₀T₁₆ C. pneumonia Ar39 A₂₄G₂₈C₂₁T₁₆pneumophila Mycoplasma A₂₃G₂₀C₁₄T₁₆ Pseudomonas putida A₂₄G₂₉C₂₁T₁₆pneumoniae Clostridium A₂₃G₂₆C₂₀T₁₉ Proteus vulgaris A₂₄G₃₀C₂₁T₁₅botulinum Enterococcus A₂₃G₂₆C₂₁T₁₈ Yersinia pestis A₂₄G₃₀C₂₁T₁₅ faeciumAcinetobacter A₂₃G₂₆C₂₁T₁₉ Yersinia A₂₄G₃₀C₂₁T₁₅ calcoacetipseudotuberculos Leptospira A₂₃G₂₆C₂₄T₁₅ Clostridium botulinumA₂₅G₂₄C₁₈T₂₁ borgpeterseni Leptospira A₂₃G₂₆C₂₄T₁₅ Clostridium tetaniA₂₅G₂₅C₁₆T₂₀ interrogans Clostridium A₂₃G₂₇C₁₉T₁₉ Francisella tularensisA₂₅G₂₅C₁₉T₁₉ perfringens Bacillus anthracis A₂₃G₂₇C₂₀T₁₈ AcinetobacterA₂₅G₂₆C₂₀T₁₉ calcoacetic Bacillus cereus A₂₃G₂₇C₂₀T₁₈ Bacteriodesfragilis A₂₅G₂₇C₁₆T₂₂ Bacillus A₂₃G₂₇C₂₀T₁₈ Chlamydophila psittaciA₂₅G₂₇C₂₁T₁₆ thuringensis Acromonas A₂₃G₂₉C₂₁T₁₆ Borrelia burgdorferiA₂₅G₂₉C₁₇T₁₉ hydrophila Escherichia coli A₂₃G₂₉C₂₁T₁₆ Streptbacillusmonilifor A₂₆G₂₆C₂₀T₁₆ Pseudomonas A₂₃G₂₉C₂₁T₁₇ Rickettsia prowazekiiA₂₆G₂₈C₁₈T₁₈ putida Escherichia coli A₂₃G₂₉C₂₂T₁₅ Rickettsia rickettsiiA₂₆G₂₈C₂₀T₁₆ Shigella A₂₃G₂₉C₂₂T₁₅ Mycoplasma mycoides A₂₈G₂₃C₁₆T₂₀dysenteriaeSome of these organisms can be distinguished using multiple primers. Forexample, Chlamydia trachomatis can be distinguished from Chlamydiapneumoniae AR39 and other organisms listed in Table II using the primer16S_(—)971-1062 and the primer 16S 1228-1310 in addition to 16S1100-1188 based on a complete or partial base composition for theamplicons.

TABLE II Organism 16S_971-1062 16S_1228-1310 16S_1100-1188 Acromonashydrophila A₂₁G₂₉C₂₂T₂₀ A₂₂G₂₇C₂₁T₁₃ A₂₃G₃₁C₂₁T₁₅ Aeromonas salmonicidaA₂₁G₂₉C₂₂T₂₀ A₂₂G₂₇C₂₁T₁₃ A₂₃G₃₁C₂₁T₁₅ Bacillus anthracis A₂₁G₂₇C₂₂T₂₂A₂₄G₂₂C₁₉T₁₈ A₂₃G₂₇C₂₀T₁₈ Bacillus cereus A₂₂G₂₇C₂₁T₂₂ A₂₄G₂₂C₁₉T₁₈A₂₃G₂₇C₂₀T₁₈ Bacillus thuringensis A₂₂G₂₇C₂₁T₂₂ A₂₄G₂₂C₁₉T₁₈A₂₃G₂₇C₂₀T₁₈ Chlamydia trachomatis A₂₂G₂₆C₂₀T₂₃ A₂₄G₂₃C₁₉T₁₆A₂₄G₂₈C₂₁T₁₆ Chlamydia pneumonia A₂₆G₂₃C₂₀T₂₂ A₂₆G₂₂C₁₆T₁₈ A₂₄G₂₈C₂₁T₁₆Ar39 Leptospira A₂₂G₂₆C₂₀T₂₁ A₂₂G₂₅C₂₁T₁₅ A₂₃G₂₆C₂₄T₁₅ borgpeterseniiLeptospira interrogans A₂₂G₂₆C₂₀T₂₁ A₂₂G₂₅C₂₁T₁₅ A₂₃G₂₆C₂₄T₁₅ Mycoplasmagenitalium A₂₈G₂₃C₁₅T₂₂ A₃₀G₁₈C₁₅T₁₉ A₂₄G₁₉C₁₂T₁₈ MycoplasmaA₂₈G₂₃C₁₅T₂₂ A₂₇G₁₉C₁₆T₂₀ A₂₃G₂₀C₁₄T₁₆ pneumoniae Escherichia coliA₂₂G₂₈C₂₀T₂₂ A₂₄G₂₅C₂₁T₁₃ A₂₃G₂₉C₂₂T₁₅ Shigella dysenteriae A₂₂G₂₈C₂₁T₂₁A₂₄G₂₅C₂₁T₁₃ A₂₃G₂₉C₂₂T₁₅ Proteus vulgaris A₂₃G₂₆C₂₂T₂₁ A₂₆G₂₄C₁₉T₁₄A₂₄G₃₀C₂₁T₁₅ Yersinia pestis A₂₄G₂₅C₂₁T₂₂ A₂₅G₂₄C₂₀T₁₄ A₂₄G₃₀C₂₁T₁₅Yersinia A₂₄G₂₅C₂₁T₂₂ A₂₅G₂₄C₂₀T₁₄ A₂₄G₃₀C₂₁T₁₅ pseudotuberculosisFrancisella tularensis A₂₀G₂₅C₂₁T₂₃ A₂₃G₂₆C₁₇T₁₇ A₂₄G₂₆C₁₉T₁₉ Rickettsiaprowazekii A₂₁G₂₆C₂₄T₂₅ A₂₄G₂₃C₁₆T₁₉ A₂₆G₂₈C₁₈T₁₈ Rickettsia rickettsiiA₂₁G₂₆C₂₅T₂₄ A₂₄G₂₄C₁₇T₁₇ A₂₆G₂₈C₂₀T₁₆

Example 2 HPLC Base Composition Determination

Example 1 can be repeated and instead of sequencing methodologies, HPLCtype methodologies can be used to determine base compositions of thevarious amplicons. Methods for determining base compositions with HPLCand UV-vis detection are described in Manderville and Kropinski,“Approaches to the Compositional Analysis of DNA,” Methods Mol Biol.2009; 502:11-7, which is herein incorporated by reference. Briefly, agiven PCR primer-pair generated amplicon can be rendered single-stranded(e.g., by heat) and then the forward primer strand or reverse primerstrand can be removed from the sample so only the sense strand oranti-sense strand is present. This can be done, for example, bysubjecting the amplicon containing sample to a solid support that hasthe forward primer linked to the solid support. Sense strands can thenhybridize to the solid support, while reverse strands are washed away.This purified sample can then digested (e.g., to completion) to generatemonodeoxynucleosides (e.g., the sample can be expose to alkalinephosphatase and a phosphodiesterase, see Huang et al., Free RadicalBiology & Medicine, 31:1341-1351, 2001, herein incorporated byreference). This sample may then be filtered to remove enzymes. Thisfiltered sample is then injected onto a HPLC column (e.g., C18 column,such as 5 um Agilent ZORBAX Exlipse XDB C18 column, 4.6 mm×150 mm) with0.1M triethylammonium acetate (pH 6.5) containing 5% CH3CN (buffer A)and 0.1M triethylammonium acetate (pH 6.5) containing 65% CH3CN (bufferB) operated at a flow rate of 1 ml/min and ambient temperature. Thissystem may employ isocratic elution with 95% buffer A and 5% buffer B.The order of elution for these conditions is dC, dG, dT, dA. Standardsfor the four deoxynucleosides should be used for comparison and togenerate standard curves that can be used for quantification. The amountof each deoxynucleoside in the single stranded amplicon is determined byintegration to give the area under each peak in the HPLC trace. Theseareas must be divided by the following extinction coefficients at 254 nmto take into account the different absorptions of the deoxynucleosidesat the detection wavelength (Connolly, 1991, “OligonucleotidesContaining Modified Bases, In F. Eckstein (Ed.), Oligonucleotides andAnalogues A Practical Approach, Oxford University Press, New York,herein incorporated by reference). Levels of” the normal nucleosides arequantified using the standard curves derived from standards. Theextinction coefficients at 254 nm: dC (6×10³), dG (13.5×10³), T (7×10³),and dA (14.3×10³). Once the base compositions are determined in thismanner, they can be compared to database entries and unknown bioagentamplicons can be identified.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, gene bankaccession numbers, internet web sites, and the like) cited in thepresent application is incorporated herein by reference in its entirety.

What is claimed is:
 1. A method of identifying a template nucleic acid,the method comprising: (a) determining at least a partial base count ofat least a subsequence of at least one template nucleic acid and/or acomplement thereof, using an approach that does not measure molecularmass of the template nucleic acid, to produce base count data; and (b)querying a database comprising at least one base count entrycorresponding to an identified nucleic acid to produce a match of thebase count data with the base count entry, thereby identifying thetemplate nucleic acid.